Plants Having Enhanced Yield-Related Traits And A Method For Making The Same

ABSTRACT

The present invention relates generally to the field of molecular biology and concerns a method for enhancing yield-related traits in plants, in particular for increasing plant yield and/or early vigour, relative to control plants. More specifically, the present invention concerns a method for enhancing yield-related traits comprising modifying the expression of a nucleic acid encoding a HAL3 polypeptide, MADS15 polypeptide, PLT transcription factor polypeptide, basic/helix-loop-helix (bHLH) transcription factor, or SPL15 transcription factor. The invention also provides constructs useful in the methods of the invention.

The present invention relates generally to the field of molecularbiology and concerns a method for improving various plant growthcharacteristics by modulating expression in a plant of a nucleic acidencoding a GRP (Growth-Related Protein). The present invention alsoconcerns plants having modulated expression of a nucleic acid encoding aGRP, which plants have improved growth characteristics relative tocorresponding wild type plants or other control plants. The inventionalso provides constructs useful in the methods of the invention.

The ever-increasing world population and the dwindling supply of arableland available for agriculture fuels research towards increasing theefficiency of agriculture. Conventional means for crop and horticulturalimprovements utilise selective breeding techniques to identify plantshaving desirable characteristics. However, such selective breedingtechniques have several drawbacks, namely that these techniques aretypically labour intensive and result in plants that often containheterogeneous genetic components that may not always result in thedesirable trait being passed on from parent plants. Advances inmolecular biology have allowed mankind to modify the germplasm ofanimals and plants. Genetic engineering of plants entails the isolationand manipulation of genetic material (typically in the form of DNA orRNA) and the subsequent introduction of that genetic material into aplant. Such technology has the capacity to deliver crops or plantshaving various improved economic, agronomic or horticultural traits.

A trait of particular economic interest is increased yield. Yield isnormally defined as the measurable produce of economic value from acrop. This may be defined in terms of quantity and/or quality. Yield isdirectly dependent on several factors, for example, the number and sizeof the organs, plant architecture (for example, the number of branches),seed production, leaf senescence and more. Root development, nutrientuptake, stress tolerance and early vigour may also be important factorsin determining yield. Optimizing the above-mentioned factors maytherefore contribute to increasing crop yield.

Seed yield is a particularly important trait, since the seeds of manyplants are important for human and animal nutrition. Crops such as corn,rice, wheat, canola and soybean account for over half the total humancaloric intake, whether through direct consumption of the seedsthemselves or through consumption of meat products raised on processedseeds. They are also a source of sugars, oils and many kinds ofmetabolites used in industrial processes. Seeds contain an embryo (thesource of new shoots and roots) and an endosperm (the source ofnutrients for embryo growth during germination and during early growthof seedlings). The development of a seed involves many genes, andrequires the transfer of metabolites from the roots, leaves and stemsinto the growing seed. The endosperm, in particular, assimilates themetabolic precursors of carbohydrates, oils and proteins and synthesizesthem into storage macromolecules to fill out the grain.

Another important trait for many crops is early vigour. Improving earlyvigour is an important objective of modern rice breeding programs inboth temperate and tropical rice cultivars. Long roots are important forproper soil anchorage in water-seeded rice. Where rice is sown directlyinto flooded fields, and where plants must emerge rapidly through water,longer shoots are associated with vigour. Where drill-seeding ispracticed, longer mesocotyls and coleoptiles are important for goodseedling emergence. The ability to engineer early vigour into plantswould be of great importance in agriculture. For example, poor earlyvigor has been a limitation to the introduction of maize (Zea mays L.)hybrids based on Corn Belt germplasm in the European Atlantic.

A further important trait is that of improved abiotic stress tolerance.Abiotic stress is a primary cause of crop loss worldwide, reducingaverage yields for most major crop plants by more than 50% (Wang et al.,Planta (2003) 218: 1-14). Abiotic stresses may be caused by drought,salinity, extremes of temperature, chemical toxicity and oxidativestress. The ability to improve plant tolerance to abiotic stress wouldbe of great economic advantage to farmers worldwide and would allow forthe cultivation of crops during adverse conditions and in territorieswhere cultivation of crops may not otherwise be possible.

Crop yield may therefore be increased by optimising one of theabove-mentioned factors.

Depending on the end use, the modification of certain yield traits maybe favoured over others. For example for applications such as forage orwood production, or bio-fuel resource, an increase in the vegetativeparts of a plant may be desirable, and for applications such as flour,starch or oil production, an increase in seed parameters may beparticularly desirable. Even amongst the seed parameters, some may befavoured over others, depending on the application. Various mechanismsmay contribute to increasing seed yield, whether that is in the form ofincreased seed size or increased seed number.

One approach to increasing yield (seed yield and/or biomass) in plantsmay be through modification of the inherent growth mechanisms of aplant, such as the cell cycle or various signalling pathways involved inplant growth or in defense mechanisms.

It has now been found that various growth characteristics may beimproved in plants by modulating expression in a plant of a nucleic acidencoding a GRP (Growth-Related Protein) in a plant. The GRP may be oneof the following: a MADS-box transcription factor (OsMADS15), a PLTtranscription factor, a bHLH transcription factor, an SPL15transcription factor, and a halotolerance protein (HAL3).

BACKGROUND Halotolerance Proteins

Many enzymatic processes in a cell require the involvement of Coenzyme A(CoA or CoASH). Coenzyme A (CoASH) itself is a highly polar molecule,consisting of adenosine 3′,5′-diphosphate linked to 4-phosphopantethenicacid (Vitamin B5) and thence to β-mercaptoethylamine. The free SH groupmay be esterified, depending on the process in which CoA is involved.The pathway of CoA synthesis has been elucidated in bacteria, animalsand plants. In plants, the conversion of the CoA precursor4′-phosphopantothenoyl-cysteine (PPC) into 4′-phosphopantetheine iscatalysed by the flavoprotein 4′-phosphopantothenoyl-cysteinedecarboxylase (PPCDC or HAL3, Kupke et al. J. Biol. Chem. 276,19190-19196, 2001). The gene encoding HAL3 proteins may be part of asmall gene family: the Arabidopsis genome comprises two isoforms(AtHAL3a and AtHAL3b; Espinoza-Ruiz et al. Plant J. 20, 529-539, 1999),in tobacco 3 HAL3 genes are present (Yonamine et al. J. Exp. Bot. 55,387-395, 2004), though in rice HAL3 is a single copy gene. It issuggested that AtHAL3 and other HAL3 proteins function as a trimer, witheach monomer having a flavin mononucleotide (FMN) bound (Albert et al.Structure 8, 961-969, 2000). The FMN cofactor is postulated to play arole in the redox reaction generating 4′-phosphopantetheine.

The molecular function of HAL3 proteins is not fully elucidated yet. TheHAL3 proteins show homology to the yeast SIS2 protein, which is involvedin halotolerance (Ferrando et al., Mol. Cell. Biol. 15, 5470-5481).Plants or plant cells ectopically expressing HAL3 show improved salt,osmotic or lithium stress tolerance (Espinoza-Ruiz et al., 1999;Yonamine et al., 2004). Tobacco HAL3a overexpression in BY2 cellsreportedly caused an increase in intracellular proline content, whichmay contribute to the salt tolerant phenotype (Yonamine et al., 2004).Furthermore, Arabidopsis plants overexpressing AtHAL3a displayed afaster growth rate than the wild type plants (Espinoza-Ruiz et al.,1999). The AtHAL3b protein was shown to interact with the cell cycleprotein CDKB1; 1 (WO 00/36124), therefore it was postulated that AtHAL3bis useful for conferring salt stress tolerance to plants and forincreasing the growth rate of plants under salt stress conditionsSurprisingly, it has now been found that preferentially increasingexpression of a nucleic acid encoding a HAL3 polypeptide in expandingtissues gives plants having increased yield relative to control plants,which yield increase is at least 10% compared to the control plants.

Transcription Factors

Transcription factors are usually defined as proteins that bind to acis-regulatory element (eg. an enhancer, a TATA box) and that, inassociation with RNA polymerase, are capable of activating and/orrepressing transcription. The Arabidopsis genome codes for at least 1533transcriptional regulators, which account for ˜5.9% of its estimatedtotal number of genes (Riechmann et al., 2000 (Science Vol. 290,2105-2109)).

MADS15

The MADS-box genes constitute a large gene family of eukaryotictranscriptional regulators involved in diverse aspects of yeast, plantand animal development. MADS-box genes encode a strongly conserved MADSdomain responsible for DNA binding to specific boxes in the regulatoryregion of their target genes. The gene family can be divided into twomain lineages, type I and type II. Type II genes are also namedMIKC-type proteins, referring to the four functional domains theypossess (FIG. 5, (Jack, Plant Mol. Biol. 46, 515-520, 2001)):

-   -   MADS for DNA binding, about 60 amino acids (highly conserved)        located at the N-terminal end of the protein;    -   I for intervening domain (less conserved), involved in the        selective formation of MADS dimer;    -   K for keratin domain (well conserved) responsible for        dimerisation;    -   C for C-terminal region (variable in sequence and length)        involved in transcriptional activation, or in the formation of a        multimeric transcription factor complex.

Over 100 MADS-box genes have been identified in Arabidopsis, and havebeen phylogenetically classified into 12 clades, each clade havingspecific deviations from the MADS consensus (Thiessen et al. J. Mol.Evol. 43, 484-516, 1996). OsMADS15 belongs to the SQUA clade (forSQUAMOSA, from Antirrhinum majus). Genes of the SQUA clade areclassified as A function organ identity genes with reference to the ABCfloral organ identity specification model, proposed by Coen andMeyerowitz in 1991 (Nature 353, 31-7). Besides OsMADS15, rice OsMADS14,OsMADS18, and OsMADS20 are also part of the SQUA clade.

The SQUA clade in dicotyledonous plants (dicots) is subdivided into twosubgroups, the AP1 and the FUL subclades (in which OsMADS15 clusters).These subclades diverge essentially with respect to the specific aminoacid motifs located at the C-terminus of their respective proteins (Littand Irish, Genetics 165, 821-833, 2003). In addition to the presence ofa specific AP1 amino acid motif, the dicot AP1 clade related proteinsusually comprise a farnesylation motif at their C-terminus (this motifis CAAX, where C is cysteine, A is usually an aliphatic amino acid, andX is methionine, glutamine, serine, cysteine or alanine). Inmonocotyledonous plants (monocots), the SQUA clade proteins are alsosubdivided into two main groups, which may be distinguished based onconserved C-terminal motifs located within the last 15 amino acids ofthe proteins: LPPWMLS (for example OsMADS15, SEQ ID NO: 117) and LPPWMLR(for example OsMADS18). In contrast to dicot sequences of the SQUAclade, monocot sequences of the SQUA clade do not possess afarnesylation motif at their C-terminus.

OsMADS15 was postulated to function in a complex with other proteins tocontrol organ formation. However, so far no mutants with a visiblephenotype have been identified; no experimental data have been presentedrelating to ectopic expression or down-regulated expression of OsMADS15,except for the data presented in WO 01/14559 (EP1209232, U.S. Pat. No.6,995,302). In this disclosure, transgenic Fagopyrum esculentum plantsexpressing OsMADS15 in sense direction showed increased branching,whereas transgenics expressing the antisense construct had decreasedgrowth and suppressed branching. Kalanchoë daigremontiana transformedwith the sense construct had leaf development around the roots, whichwas taken as an indication of increased branching. The authors statedthat no changes were observed in terms of flower development andstructure of these flowers. The orthologue of OsMADS15 in Arabidopsis isAPETALA1 (AP1). Ectopic expression of AP1 results in a decrease offlowering time, and AP1 mutants exhibit delayed flowering and haveabnormal flowers.

PLT

The AP2 (apetala2)/ERF (ethylene-responsive responsive element-bindingfactor) family comprises transcription factors with at least one highlyconserved DNA binding domain, the AP2 domain. The AP2 domain wasoriginally described in APETALA2 (AP2), an Arabidopsis protein involvedin developmental programs such as meristem identity regulation andfloral organ specification (Jofuku et al., (1994) Plant Cell 6,1211-1225). AP2/ERF proteins are divided into subfamilies based onwhether they contain one (ERF subfamily) or two (AP2 subfamily) DNAbinding domains (FIG. 12). More than 140 AP2/ERF genes have beenidentified in the Arabidopsis thaliana genome (Reichmann et al. (2000)Science 290: 2105-2110), amongst which up to 18 belong to the AP2subfamily (Kim et al. (2006) Mol Bio Evol 23(1): 107-120).

Two Arabidopsis AP2 subfamily transcription factor polypeptides, encodedby the Plethora 1 (PLT1) and Plethora 2 (PLT2) genes, collectivelycalled PLT genes, have been shown to be required for stem cellspecification and maintenance specifically in the root meristem. InRT-PCR analysis, PLT transcripts were mainly detected in roots,indicating that PLT expression is strongly associated with rootidentity. Ectopic PLT expression in the Arabidopsis embryo induceshomeotic transformation of apical domains into root stem cells, roots,or hypocotyls (Aida et al (2004) Cell 119:109-120).

US patent application 2004/0045049 and international patent applicationWO03/013227 provide the nucleic acid sequence encoding the ArabidopsisPLT1 transcription factor (referred to in the applications as G1793).Overexpression of G1793 (using the CaMV 35S promoter) in Arabidopsis wasreported to produce alterations in cotyledon morphology, a mildreduction in overall plant size and thin inflorescences (possibly withabnormal flowers) compared to wild type controls. G1793 overexpressorsproduced more seed oil than control plants. Nucleic acid sequencesencoding potential Glycine max, Oryza sativa and Zea mays orthologs areprovided.

International patent application WO 03/002751 provides for a Glycine maxnucleic acid sequence encoding a PLT polypeptide, with sequencesimilarity to a corn nucleic acid sequence identified by gene profiling(by DNA microarray).

International patent application WO 05/075655 provides for Oryza sativaand Zea mays nucleic acid sequences with sequence similarity to theArabidopsis PLT genes.

bHLH

The basic/helix-loop-helix (bHLH) proteins are a superfamily oftranscription factors that bind as dimers to specific DNA target sitesand that have been well characterized in non-plant eukaryotes asimportant regulatory components in diverse biological processes. Thedistinguishing characteristic of the bHLH family is a bipartite domainconsisting of approximately 60 amino acids. This bipartite domain iscomprised of a DNA-binding basic region, which binds to a consensushexanucleotide E-box and two α-helices separated by a variable loopregion. The two α-helices promote dimerization, allowing the formationof homo- and heterodimers between different family members. While thebHLH domain is evolutionarily conserved, there is little sequencesimilarity between clades beyond the domain.

Bailey et al., 2003 (The Plant Cell, Vol. 15, 2497-2501) report thetotal number of detected bHLH genes in Arabidopsis thaliana to be 162,making bHLH genes one of the largest families of transcription factorsin Arabidopsis; the rice genome reportedly contains 131 bHLH genes (Buckand Atchley, 2003 (J. Mol. Evol. 56:742-750)). Heim et al., 2003 (Mol.Biol. Evol. 20(5):735-747) identified 12 subfamilies of bHLH genes fromArabidopsis thaliana based on structural similarities.

The bHLH proteins from plants that have been characterized have beenreported to function in anthocyanin biosynthesis, phytochrome signaling,globulin expression, fruit dehiscence, carpel and epidermal development(Buck and Atchley, 2003).

SPL

The Squamosa promoter binding protein-like (SPL) transcription factorpolypeptides are structurally diverse proteins that share a highlyconserved DNA binding domain (DBD) of about 80 amino acid residues inlength (Klein et al. (1996) Mol Gen Genet 259: 7-16; Cardon et al.(1999) Gene 237: 91-104). The SPL transcription factor DNA consensussequence binding site in the promoter of target genes is 5′-TNCGTACAA-3′where N represents any base. Within the SPL DBD are ten conservedcysteine (Cys) or histidine (His) residues (see FIG. 23) of which eightare zinc coordinating residues binding two zinc ions necessary for theformation of SPL specific zinc finger tertiary structure (Yamasaki etal. (2004) J Mol Biol 337: 49-63). A second conserved feature within theSPL DBD is a bipartite nuclear localisation signal. Outside of the DBD,a micro RNA (miRNA) target motif (miR156) is found in most of thenucleic acid sequences encoding SPL transcription factor polypeptides(either in the coding region, or the 3′ UTR) across the plant kingdom(Rhoades et al. (2002) Cell 110: 513-520). miRNAs control SPL geneexpression post-transcriptionally by targeting SPL encoding mRNAs fordegradation or by translational repression.

Riechmann et al. (Science 290: 2105-2109, 2000) report 16 SPLtranscription factor polypeptides in Arabidopsis thaliana, with littlesequence similarity between themselves (apart from the abovementionedfeatures), the size of the deduced SPL polypeptide ranging from 131 to927 amino acids. Nevertheless, pairs of SPL transcription factorpolypeptides sharing higher sequence homology were detected within theSPL family of this plant (Cardon et al. (1999)).

The SPL transcription factor polypeptides (only found in plants)characterized to date have been shown to function in plant development,in particular in flower development. Transgenic plants overexpressing anSPL3 transcription factor polypeptide were reported to flower earlier(Cardon et al. (1997) Plant J 12: 367-377).

In European patent application EP1033405, the nucleic acid and deducedpolypeptide sequences of the SPL15 transcription factor polypeptide arepresented.

In international patent application WO03013227, a nucleic acid sequence(and deduced polypeptide sequence; internal reference G2346) ispresented that encodes part of the SPL15 transcription factorpolypeptide however 38 amino acids from the C-terminal end of the SPL15transcription factor. Transgenic Arabidopsis thaliana plantsconstitutively overexpressing the modified SPL15 transcription factor(or G2346) polypeptide have slightly enlarged cotyledons. At laterstages of development, the same plants are reported to show noconsistent differences from control plants.

SUMMARY OF THE INVENTION

Surprisingly, it has now been found that preferentially increasingexpression of a nucleic acid encoding a HAL3 polypeptide in expandingtissues gives plants having enhanced yield-related traits, in particularincreased early vigour and increased seed yield relative to controlplants, which seed yield increase is at least 10% compared to thecontrol plants.

According to another embodiment of present invention, there is provideda method for enhancing yield-related traits, in particular forincreasing early vigour of a plant and increasing seed yield of a plant,relative to control plants, comprising preferentially increasingexpression of a nucleic acid encoding a HAL3 polypeptide in expandingtissues of a plant.

Surprisingly, it has now been found that modulating expression of anucleic acid encoding a MADS15 polypeptide gives plants having enhancedyield-related traits, in particular increased yield relative to controlplants.

According one embodiment, there is provided a method for increasingplant yield relative to control plants, comprising increasing expressionof a nucleic acid encoding a MADS15 polypeptide in a plant. Theincreased yield comprised increased vegetative biomass but not increasedseed yield.

According to another embodiment, the present invention provides methodsfor increasing yield of a plant relative to control plants, comprisingdecreasing the level and/or activity of an endogenous MADS15polypeptide. The increased yield comprised higher seed yield, but didnot comprise increased vegetative biomass.

Surprisingly, it has now been found that increasing expression of anucleic acid encoding a PLT transcription factor polypeptide givesplants having enhanced yield related traits, in particular increasedyield relative to control plants.

According to a further embodiment, there is provided a method forincreasing plant yield relative to control plants, comprising increasingexpression in a plant of a nucleic acid encoding a PLT transcriptionfactor polypeptide.

Surprisingly, it has now been found that modulating expression in aplant of a nucleic acid encoding a particular class of bHLHtranscription factor gives plants having enhanced yield-related traitsrelative to control plants. The particular class of bHLH transcriptionfactor suitable for enhancing yield-related traits in plants isdescribed in detail below.

According to a further embodiment, the present invention provides amethod for enhancing yield-related traits in plants relative to controlplants, comprising modulating expression in a plant of a nucleic acidencoding a particular class of bHLH transcription factor.

Surprisingly, it has now been found that increasing expression in aplant of a nucleic acid encoding an SPL15 transcription factorpolypeptide gives plants having enhanced yield related traits, inparticular increased yield relative to control plants.

According to a further embodiment, the invention provides a method forincreasing yield in plants relative to control plants, comprisingincreasing expression in a plant of a nucleic acid encoding an SPL15transcription factor polypeptide.

DEFINITIONS Polypeptide(s)/Protein(s)

The terms “polypeptide” and “protein” are used interchangeably hereinand refer to amino acids in a polymeric form of any length.

Polynucleotide(s)/Nucleic Acid(s)/Nucleic Acid Sequence(s)/NucleotideSequence(s)

The terms “polynucleotide(s)”, “nucleic acid sequence(s)”, “nucleotidesequence(s)”, “nucleic acid(s)” are used interchangeably herein andrefer to nucleotides, either ribonucleotides or deoxyribonucleotides ora combination of both, in a polymeric form of any length.

Control Plant(s)

The choice of suitable control plants is a routine part of anexperimental setup and may include corresponding wild type plants orcorresponding plants without the gene of interest. The control plant istypically of the same plant species or even of the same variety as theplant to be assessed. The control plant may also be a nullizygote of theplant to be assessed. A “control plant” as used herein refers not onlyto whole plants, but also to plant parts, including seeds and seedparts.

Homoloque(s)

“Homologues” of a protein encompass peptides, oligopeptides,polypeptides, proteins and enzymes having amino acid substitutions,deletions and/or insertions relative to the unmodified protein inquestion and having similar biological and functional activity as theunmodified protein from which they are derived.

A deletion refers to removal of one or more amino acids from a protein.

An insertion refers to one or more amino acid residues being introducedinto a predetermined site in a protein. Insertions may compriseN-terminal and/or C-terminal fusions as well as intra-sequenceinsertions of single or multiple amino acids. Generally, insertionswithin the amino acid sequence will be smaller than N- or C-terminalfusions, of the order of about 1 to 10 residues. Examples of N- orC-terminal fusion proteins or peptides include the binding domain oractivation domain of a transcriptional activator as used in the yeasttwo-hybrid system, phage coat proteins, (histidine)-6-tag, glutathioneS-transferase-tag, protein A, maltose-binding protein, dihydrofolatereductase, Tag•100 epitope, c-myc epitope, FLAG®-epitope, lacZ, CMP(calmodulin-binding peptide), HA epitope, protein C epitope and VSVepitope.

A substitution refers to replacement of amino acids of the protein withother amino acids having similar properties (such as similarhydrophobicity, hydrophilicity, antigenicity, propensity to form orbreak α-helical structures or β-sheet structures). Amino acidsubstitutions are typically of single residues, but may be clustereddepending upon functional constraints placed upon the polypeptide;insertions will usually be of the order of about 1 to 10 amino acidresidues. The amino acid substitutions are preferably conservative aminoacid substitutions. Conservative substitution tables are well known inthe art (see for example Creighton (1984) Proteins. W.H. Freeman andCompany and Table 1 below).

TABLE 1 Examples of conserved amino acid substitutions ConservativeConservative Residue Substitutions Residue Substitutions Ala Ser LeuIle; Val Arg Lys Lys Arg; Gln Asn Gln; His Met Leu; Ile Asp Glu Phe Met;Leu; Tyr Gln Asn Ser Thr; Gly Cys Ser Thr Ser; Val Glu Asp Trp Tyr GlyPro Tyr Trp; Phe His Asn; Gln Val Ile; Leu Ile Leu, Val

Amino acid substitutions, deletions and/or insertions may readily bemade using peptide synthetic techniques well known in the art, such assolid phase peptide synthesis and the like, or by recombinant DNAmanipulation. Methods for the manipulation of DNA sequences to producesubstitution, insertion or deletion variants of a protein are well knownin the art. For example, techniques for making substitution mutations atpredetermined sites in DNA are well known to those skilled in the artand include M13 mutagenesis, T7-Gen in vitro mutagenesis (USB,Cleveland, Ohio), QuickChange Site Directed mutagenesis (Stratagene, SanDiego, Calif.), PCR-mediated site-directed mutagenesis or othersite-directed mutagenesis protocols.

Derivatives

“Derivatives” include peptides, oligopeptides, polypeptides which may,compared to the amino acid sequence of the naturally-occurring form ofthe protein, such as the protein of interest, comprise substitutions ofamino acids with non-naturally occurring amino acid residues, oradditions of non-naturally occurring amino acid residues. “Derivatives”of a protein also encompass peptides, oligopeptides, polypeptides whichcomprise naturally occurring altered (glycosylated, acylated,prenylated, phosphorylated, myristoylated, sulphated etc.) ornon-naturally altered amino acid residues compared to the amino acidsequence of a naturally-occurring form of the polypeptide. A derivativemay also comprise one or more non-amino acid substituents or additionscompared to the amino acid sequence from which it is derived, forexample a reporter molecule or other ligand, covalently ornon-covalently bound to the amino acid sequence, such as a reportermolecule which is bound to facilitate its detection, and non-naturallyoccurring amino acid residues relative to the amino acid sequence of anaturally-occurring protein.

Orthologue(s)/Paralogue(s)

Orthologues and paralogues encompass evolutionary concepts used todescribe the ancestral relationships of genes. Paralogues are geneswithin the same species that have originated through duplication of anancestral gene and orthologues are genes from different organisms thathave originated through speciation.

Domain

The term “domain” refers to a set of amino acids conserved at specificpositions along an alignment of sequences of evolutionarily relatedproteins. While amino acids at other positions can vary betweenhomologues, amino acids that are highly conserved at specific positionsindicate amino acids that are likely essential in the structure,stability or function of a protein. Identified by their high degree ofconservation in aligned sequences of a family of protein homologues,they can be used as identifiers to determine if any polypeptide inquestion belongs to a previously identified polypeptide family.

Motif/Consensus Sequence/Signature

The term “motif” or “consensus sequence” or “signature” refers to ashort conserved region in the sequence of evolutionarily relatedproteins. Motifs are frequently highly conserved parts of domains, butmay also include only part of the domain, or be located outside ofconserved domain (if all of the amino acids of the motif fall outside ofa defined domain).

Hybridisation

The term “hybridisation” as defined herein is a process whereinsubstantially homologous complementary nucleotide sequences anneal toeach other. The hybridisation process can occur entirely in solution,i.e. both complementary nucleic acids are in solution. The hybridisationprocess can also occur with one of the complementary nucleic acidsimmobilised to a matrix such as magnetic beads, Sepharose beads or anyother resin. The hybridisation process can furthermore occur with one ofthe complementary nucleic acids immobilised to a solid support such as anitro-cellulose or nylon membrane or immobilised by e.g.photolithography to, for example, a siliceous glass support (the latterknown as nucleic acid arrays or microarrays or as nucleic acid chips).In order to allow hybridisation to occur, the nucleic acid molecules aregenerally thermally or chemically denatured to melt a double strand intotwo single strands and/or to remove hairpins or other secondarystructures from single stranded nucleic acids.

The term “stringency” refers to the conditions under which ahybridisation takes place. The stringency of hybridisation is influencedby conditions such as temperature, salt concentration, ionic strengthand hybridisation buffer composition. Generally, low stringencyconditions are selected to be about 30° C. lower than the thermalmelting point (T_(m)) for the specific sequence at a defined ionicstrength and pH. Medium stringency conditions are when the temperatureis 20° C. below T_(m), and high stringency conditions are when thetemperature is 10° C. below T_(m). High stringency hybridisationconditions are typically used for isolating hybridising sequences thathave high sequence similarity to the target nucleic acid sequence.However, nucleic acids may deviate in sequence and still encode asubstantially identical polypeptide, due to the degeneracy of thegenetic code. Therefore medium stringency hybridisation conditions maysometimes be needed to identify such nucleic acid molecules.

The T_(m) is the temperature under defined ionic strength and pH, atwhich 50% of the target sequence hybridises to a perfectly matchedprobe. The T_(m) is dependent upon the solution conditions and the basecomposition and length of the probe. For example, longer sequenceshybridise specifically at higher temperatures. The maximum rate ofhybridisation is obtained from about 16° C. up to 32° C. below T_(m).The presence of monovalent cations in the hybridisation solution reducethe electrostatic repulsion between the two nucleic acid strands therebypromoting hybrid formation; this effect is visible for sodiumconcentrations of up to 0.4M (for higher concentrations, this effect maybe ignored). Formamide reduces the melting temperature of DNA-DNA andDNA-RNA duplexes with 0.6 to 0.7° C. for each percent formamide, andaddition of 50% formamide allows hybridisation to be performed at 30 to45° C., though the rate of hybridisation will be lowered. Base pairmismatches reduce the hybridisation rate and the thermal stability ofthe duplexes. On average and for large probes, the T_(m) decreases about1° C. per % base mismatch. The T_(m) may be calculated using thefollowing equations, depending on the types of hybrids:

1) DNA-DNA hybrids (Meinkoth and Wahl, Anal. Biochem., 138: 267-284,1984):

T _(m)=81.5° C.+16.6×log₁₀[Na⁺]^(a)+0.41×%[G/C ^(b)]−500×[L^(c)]⁻¹−0.61×% formamide

2) DNA-RNA or RNA-RNA hybrids:

T _(m)=79.8+18.5(log₁₀[Na⁺]^(a))+0.58(%G/C ^(b))+11.8 (%G/C ^(b))²−820/L^(c)

3) oligo-DNA or oligo-RNA^(d) hybrids:For <20 nucleotides: T_(m)=22 (I_(n))For 20-35 nucleotides: T_(m)=22+1.46 (I_(n))

-   -   ^(a) or for other monovalent cation, but only accurate in the        0.01-0.4 M range.    -   ^(b) only accurate for % GC in the 30% to 75% range.    -   ^(c) L=length of duplex in base pairs.    -   ^(d) Oligo, oligonucleotide; I_(n), effective length of        primer=2×(no. of G/C)+(no. of NT).

Non-specific binding may be controlled using any one of a number ofknown techniques such as, for example, blocking the membrane withprotein containing solutions, additions of heterologous RNA, DNA, andSDS to the hybridisation buffer, and treatment with Rnase. Fornon-homologous probes, a series of hybridizations may be performed byvarying one of (i) progressively lowering the annealing temperature (forexample from 68° C. to 42° C.) or (ii) progressively lowering theformamide concentration (for example from 50% to 0%). The skilledartisan is aware of various parameters which may be altered duringhybridisation and which will either maintain or change the stringencyconditions.

Besides the hybridisation conditions, specificity of hybridisationtypically also depends on the function of post-hybridisation washes. Toremove background resulting from non-specific hybridisation, samples arewashed with dilute salt solutions. Critical factors of such washesinclude the ionic strength and temperature of the final wash solution:the lower the salt concentration and the higher the wash temperature,the higher the stringency of the wash. Wash conditions are typicallyperformed at or below hybridisation stringency. A positive hybridisationgives a signal that is at least twice of that of the background.Generally, suitable stringent conditions for nucleic acid hybridisationassays or gene amplification detection procedures are as set forthabove. More or less stringent conditions may also be selected. Theskilled artisan is aware of various parameters which may be alteredduring washing and which will either maintain or change the stringencyconditions.

For example, typical high stringency hybridisation conditions for DNAhybrids longer than 50 nucleotides encompass hybridisation at 65° C. in1×SSC or at 42° C. in 1×SSC and 50% formamide, followed by washing at65° C. in 0.3×SSC. Examples of medium stringency hybridisationconditions for DNA hybrids longer than 50 nucleotides encompasshybridisation at 50° C. in 4×SSC or at 40° C. in 6×SSC and 50%formamide, followed by washing at 50° C. in 2×SSC. The length of thehybrid is the anticipated length for the hybridising nucleic acid. Whennucleic acids of known sequence are hybridised, the hybrid length may bedetermined by aligning the sequences and identifying the conservedregions described herein. 1×SSC is 0.15M NaCl and 15 mM sodium citrate;the hybridisation solution and wash solutions may additionally include5×Denhardt's reagent, 0.5-1.0% SDS, 100 μg/ml denatured, fragmentedsalmon sperm DNA, 0.5% sodium pyrophosphate.

For the purposes of defining the level of stringency, reference can bemade to Sambrook et al. (2001) Molecular Cloning: a laboratory manual,3rd Edition Cold Spring Harbor Laboratory Press, CSH, New York or toCurrent Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989and yearly updates).

Splice Variant

The term “splice variant” as used herein encompasses variants of anucleic acid sequence in which selected introns and/or exons have beenexcised, replaced, displaced or added, or in which introns have beenshortened or lengthened. Such variants will be ones in which thebiological activity of the protein is substantially retained; this maybe achieved by selectively retaining functional segments of the protein.Such splice variants may be found in nature or may be manmade. Methodsfor predicting and isolating such splice variants are well known in theart (see for example Foissac and Schiex, BMC Bioinformatics. 2005; 6:25).

Allelic Variant

Alleles or allelic variants are alternative forms of a given gene,located at the same chromosomal position. Allelic variants encompassSingle Nucleotide Polymorphisms (SNPs), as well as SmallInsertion/Deletion Polymorphisms (INDELs). The size of INDELs is usuallyless than 100 bp. SNPs and INDELs form the largest set of sequencevariants in naturally occurring polymorphic strains of most organisms.

Gene Shuffling/Directed Evolution

Gene shuffling or directed evolution consists of iterations of DNAshuffling followed by appropriate screening and/or selection to generatevariants of nucleic acids or portions thereof encoding proteins having amodified biological activity (Castle et al., (2004) Science 304(5674):1151-4; U.S. Pat. Nos. 5,811,238 and 6,395,547).

Regulatory Element/Control Sequence/Promoter

The terms “regulatory element”, “control sequence” and “promoter” areall used interchangeably herein and are to be taken in a broad contextto refer to regulatory nucleic acid sequences capable of effectingexpression of the sequences to which they are ligated. The term“promoter” typically refers to a nucleic acid control sequence locatedupstream from the transcriptional start of a gene and which is involvedin recognising and binding of RNA polymerase and other proteins, therebydirecting transcription of an operably linked nucleic acid. Encompassedby the aforementioned terms are transcriptional regulatory sequencesderived from a classical eukaryotic genomic gene (including the TATA boxwhich is required for accurate transcription initiation, with or withouta CCAAT box sequence) and additional regulatory elements (i.e. upstreamactivating sequences, enhancers and silencers) which alter geneexpression in response to developmental and/or external stimuli, or in atissue-specific manner. Also included within the term is atranscriptional regulatory sequence of a classical prokaryotic gene, inwhich case it may include a −35 box sequence and/or −10 boxtranscriptional regulatory sequences. The term “regulatory element” alsoencompasses a synthetic fusion molecule or derivative that confers,activates or enhances expression of a nucleic acid molecule in a cell,tissue or organ.

A “plant promoter” comprises regulatory elements, which mediate theexpression of a coding sequence segment in plant cells. Accordingly, aplant promoter need not be of plant origin, but may originate fromviruses or micro-organisms, for example from viruses which attack plantcells. The “plant promoter” can also originate from a plant cell, e.g.from the plant which is transformed with the nucleic acid sequence to beexpressed in the inventive process and described herein. This alsoapplies to other “plant” regulatory signals, such as “plant”terminators. The promoters upstream of the nucleotide sequences usefulin the methods of the present invention can be modified by one or morenucleotide substitution(s), insertion(s) and/or deletion(s) withoutinterfering with the functionality or activity of either the promoters,the open reading frame (ORF) or the 3′-regulatory region such asterminators or other 3′ regulatory regions which are located away fromthe ORF. It is furthermore possible that the activity of the promotersis increased by modification of their sequence, or that they arereplaced completely by more active promoters, even promoters fromheterologous organisms. For expression in plants, the nucleic acidmolecule must, as described above, be linked operably to or comprise asuitable promoter which expresses the gene at the right point in timeand with the required spatial expression pattern.

For the identification of functionally equivalent promoters, thepromoter strength and/or expression pattern of a candidate promoter maybe analysed for example by operably linking the promoter to a reportergene and assaying the expression level and pattern of the reporter genein various tissues of the plant. Suitable well-known reporter genesinclude for example beta-glucuronidase or beta galactosidase. Thepromoter activity is assayed by measuring the enzymatic activity of thebeta-glucuronidase or beta-galactosidase. The promoter strength and/orexpression pattern may then be compared to that of a reference promoter(such as the one used in the methods of the present invention).Alternatively, promoter strength may be assayed by quantifying mRNAlevels or by comparing mRNA levels of the nucleic acid used in themethods of the present invention, with mRNA levels of housekeeping genessuch as 18S rRNA, using methods known in the art, such as Northernblotting with densitometric analysis of autoradiograms, quantitativereal-time PCR or RT-PCR (Heid et al., 1996 Genome Methods 6: 986-994).Generally by “weak promoter” is intended a promoter that drivesexpression of a coding sequence at a low level. By “low level” isintended at levels of about 1/10,000 transcripts to about 1/100,000transcripts, to about 1/500,0000 transcripts per cell. Conversely, a“strong promoter” drives expression of a coding sequence at high level,or at about 1/10 transcripts to about 1/100 transcripts to about 1/1,000transcripts per cell.

Operably Linked

The term “operably linked” as used herein refers to a functional linkagebetween the promoter sequence and the gene of interest, such that thepromoter sequence is able to initiate transcription of the gene ofinterest.

Constitutive Promoter

A “constitutive promoter” refers to a promoter that is transcriptionallyactive during most, but not necessarily all, phases of growth anddevelopment and under most environmental conditions, in at least onecell, tissue or organ. Table 2 below gives examples of constitutivepromoters.

TABLE 2 Examples of constitutive promoters Gene Source Reference ActinMcElroy et al, Plant Cell, 2: 163-171, 1990 HMGB WO 2004/070039 CAMV 35SOdell et al, Nature, 313: 810-812, 1985 CaMV 19S Nilsson et al.,Physiol. Plant. 100: 456-462, 1997 GOS2 de Pater et al, Plant J Nov;2(6): 837-44, 1992, WO 2004/065596 Ubiquitin Christensen et al, PlantMol. Biol. 18: 675-689, 1992 Rice cyclophilin Buchholz et al, Plant MolBiol. 25(5): 837-43, 1994 Maize H3 histone Lepetit et al, Mol. Gen.Genet. 231: 276-285, 1992 Alfalfa H3 histone Wu et al. Plant Mol. Biol.11: 641-649, 1988 Actin 2 An et al, Plant J. 10(1); 107-121, 1996 34SFMV Sanger et al., Plant. Mol. Biol., 14, 1990: 433-443 Rubisco U.S.Pat. No. 4,962,028 small subunit OCS Leisner (1988) Proc Natl Acad SciUSA 85(5): 2553 SAD1 Jain et al., Crop Science, 39 (6), 1999: 1696 SAD2Jain et al., Crop Science, 39 (6), 1999: 1696 nos Shaw et al. (1984)Nucleic Acids Res. 12(20): 7831-7846 V-ATPase WO 01/14572 Super promoterWO 95/14098 G-box proteins WO 94/12015

Ubiquitous Promoter

A ubiquitous promoter is active in substantially all tissues or cells ofan organism.

Developmentally-Regulated Promoter

A developmentally-regulated promoter is active during certaindevelopmental stages or in parts of the plant that undergo developmentalchanges.

Inducible Promoter

An inducible promoter has induced or increased transcription initiationin response to a chemical (for a review see Gatz 1997, Annu. Rev. PlantPhysiol. Plant Mol. Biol., 48:89-108), environmental or physicalstimulus, or may be “stress-inducible”, i.e. activated when a plant isexposed to various stress conditions, or a “pathogen-inducible” i.e.activated when a plant is exposed to exposure to various pathogens.

Organ-Specific/Tissue-Specific Promoter

An organ-specific or tissue-specific promoter is one that is capable ofpreferentially initiating transcription in certain organs or tissues,such as the leaves, roots, seed tissue etc. For example, a“root-specific promoter” is a promoter that is transcriptionally activepredominantly in plant roots, substantially to the exclusion of anyother parts of a plant, whilst still allowing for any leaky expressionin these other plant parts. Promoters able to initiate transcription incertain cells only are referred to herein as “cell-specific”.

A green tissue-specific promoter as defined herein is a promoter that istranscriptionally active predominantly in green tissue, substantially tothe exclusion of any other parts of a plant, whilst still allowing forany leaky expression in these other plant parts.

Examples of green tissue-specific promoters which may be used to performthe methods of the invention are shown in Table 3 below.

TABLE 3 Examples of green tissue-specific promoters Gene ExpressionReference Maize Orthophosphate dikinase Leaf specific Fukavama et al.,2001 Maize Phosphoenolpyruvate Leaf specific Kausch et al., 2001carboxylase Rice Phosphoenolpyruvate Leaf specific Liu et al., 2003carboxylase Rice small subunit Rubisco Leaf specific Nomura et al., 2000rice beta expansin EXBP9 Shoot specific WO 2004/070039 Pigeonpea smallsubunit Leaf specific Panguluri et al., 2005 Rubisco Pea RBCS3A Leafspecific

Another example of a tissue-specific promoter is a meristem-specificpromoter, which is transcriptionally active predominantly inmeristematic tissue, substantially to the exclusion of any other partsof a plant, whilst still allowing for any leaky expression in theseother plant parts

Terminator

The term “terminator” encompasses a control sequence which is a DNAsequence at the end of a transcriptional unit which signals 3′processing and polyadenylation of a primary transcript and terminationof transcription. The terminator can be derived from the natural gene,from a variety of other plant genes, or from T-DNA. The terminator to beadded may be derived from, for example, the nopaline synthase oroctopine synthase genes, or alternatively from another plant gene, orless preferably from any other eukaryotic gene.

Modulation

The term “modulation” means in relation to expression or geneexpression, a process in which the expression level is changed by saidgene expression in comparison to the control plant, preferably theexpression level is increased. The original, unmodulated expression maybe of any kind of expression of a structural RNA (rRNA, tRNA) or mRNAwith subsequent translation. The term “modulating the activity” shallmean any change of the expression of the inventive nucleic acidsequences or encoded proteins, which leads to increased yield and/orincreased growth of the plants.

Increased Expression/Overexpression

The term “increased expression” or “overexpression” as used herein meansany form of expression that is additional to the original wild-typeexpression level.

Methods for increasing expression of genes or gene products are welldocumented in the art and include, for example, overexpression driven byappropriate promoters, the use of transcription enhancers or translationenhancers. Isolated nucleic acids which serve as promoter or enhancerelements may be introduced in an appropriate position (typicallyupstream) of a non-heterologous form of a polynucleotide so as toupregulate expression of a nucleic acid encoding the polypeptide ofinterest. For example, endogenous promoters may be altered in vivo bymutation, deletion, and/or substitution (see, Kmiec, U.S. Pat. No.5,565,350; Zarling et al., PCT/US93/03868), or isolated promoters may beintroduced into a plant cell in the proper orientation and distance froma gene of the present invention so as to control the expression of thegene.

If polypeptide expression is desired, it is generally desirable toinclude a polyadenylation region at the 3′-end of a polynucleotidecoding region. The polyadenylation region can be derived from thenatural gene, from a variety of other plant genes, or from T-DNA. The 3′end sequence to be added may be derived from, for example, the nopalinesynthase or octopine synthase genes, or alternatively from another plantgene, or less preferably from any other eukaryotic gene.

An intron sequence may also be added to the 5′ untranslated region (UTR)or the coding sequence of the partial coding sequence to increase theamount of the mature message that accumulates in the cytosol. Inclusionof a spliceable intron in the transcription unit in both plant andanimal expression constructs has been shown to increase gene expressionat both the mRNA and protein levels up to 1000-fold (Buchman and Berg(1988) Mol. Cell biol. 8: 4395-4405; Callis et al. (1987) Genes Dev1:1183-1200). Such intron enhancement of gene expression is typicallygreatest when placed near the 5′ end of the transcription unit. Use ofthe maize introns Adh1-S intron 1, 2, and 6, the Bronze-1 intron areknown in the art. For general information see: The Maize Handbook,Chapter 116, Freeling and Walbot, Eds., Springer, N.Y. (1994).

Endogenous Gene

Reference herein to an “endogenous” gene not only refers to the gene inquestion as found in a plant in its natural form (i.e., without therebeing any human intervention), but also refers to that same gene (or asubstantially homologous nucleic acid/gene) in an isolated formsubsequently (re)introduced into a plant (a transgene). For example, atransgenic plant containing such a transgene may encounter a substantialreduction of the transgene expression and/or substantial reduction ofexpression of the endogenous gene.

Decreased Expression

Reference herein to “reduction or substantial elimination” is taken tomean a decrease in endogenous gene expression and/or polypeptide levelsand/or polypeptide activity relative to control plants. The reduction orsubstantial elimination is in increasing order of preference at least10%, 20%, 30%, 40% or 50%, 60%, 70%, 80%, 85%, 90%, or 95%, 96%, 97%,98%, 99% or more reduced compared to that of control plants.

For the reduction or substantial elimination of expression an endogenousgene in a plant, a sufficient length of substantially contiguousnucleotides of a nucleic acid sequence is required. In order to performgene silencing, this may be as little as 20, 19, 18, 17, 16, 15, 14, 13,12, 11, 10 or fewer nucleotides, alternatively this may be as much asthe entire gene (including the 5′ and/or 3′ UTR, either in part or inwhole). The stretch of substantially contiguous nucleotides may bederived from the nucleic acid encoding the protein of interest (targetgene), or from any nucleic acid capable of encoding an orthologue,paralogue or homologue of the protein of interest. Preferably, thestretch of substantially contiguous nucleotides is capable of forminghydrogen bonds with the target gene (either sense or antisense strand),more preferably, the stretch of substantially contiguous nucleotideshas, in increasing order of preference, 50%, 60%, 70%, 80%, 85%, 90%,95%, 96%, 97%, 98%, 99%, 100% sequence identity to the target gene(either sense or antisense strand). A nucleic acid sequence encoding a(functional) polypeptide is not a requirement for the various methodsdiscussed herein for the reduction or substantial elimination ofexpression of an endogenous gene.

This reduction or substantial elimination of expression may be achievedusing routine tools and techniques. A preferred method for the reductionor substantial elimination of endogenous gene expression is byintroducing and expressing in a plant a genetic construct into which thenucleic acid (in this case a stretch of substantially contiguousnucleotides derived from the gene of interest, or from any nucleic acidcapable of encoding an orthologue, paralogue or homologue of any one ofthe protein of interest) is cloned as an inverted repeat (in part orcompletely), separated by a spacer (non-coding DNA).

In such a preferred method, expression of the endogenous gene is reducedor substantially eliminated through RNA-mediated silencing using aninverted repeat of a nucleic acid or a part thereof (in this case astretch of substantially contiguous nucleotides derived from the gene ofinterest, or from any nucleic acid capable of encoding an orthologue,paralogue or homologue of the protein of interest), preferably capableof forming a hairpin structure. The inverted repeat is cloned in anexpression vector comprising control sequences. A non-coding DNA nucleicacid sequence (a spacer, for example a matrix attachment region fragment(MAR), an intron, a polylinker, etc.) is located between the twoinverted nucleic acids forming the inverted repeat. After transcriptionof the inverted repeat, a chimeric RNA with a self-complementarystructure is formed (partial or complete). This double-stranded RNAstructure is referred to as the hairpin RNA (hpRNA). The hpRNA isprocessed by the plant into siRNAs that are incorporated into anRNA-induced silencing complex (RISC). The RISC further cleaves the mRNAtranscripts, thereby substantially reducing the number of mRNAtranscripts to be translated into polypeptides. For further generaldetails see for example, Grierson et al. (1998) WO 98/53083; Waterhouseet al. (1999) WO 99/53050).

Performance of the methods of the invention does not rely on introducingand expressing in a plant a genetic construct into which the nucleicacid is cloned as an inverted repeat, but any one or more of severalwell-known “gene silencing” methods may be used to achieve the sameeffects.

One such method for the reduction of endogenous gene expression isRNA-mediated silencing of gene expression (downregulation). Silencing inthis case is triggered in a plant by a double stranded RNA sequence(dsRNA) that is substantially similar to the target endogenous gene.This dsRNA is further processed by the plant into about 20 to about 26nucleotides called short interfering RNAs (siRNAs). The siRNAs areincorporated into an RNA-induced silencing complex (RISC) that cleavesthe mRNA transcript of the endogenous target gene, thereby substantiallyreducing the number of mRNA transcripts to be translated into apolypeptide. Preferably, the double stranded RNA sequence corresponds toa target gene.

Another example of an RNA silencing method involves the introduction ofnucleic acid sequences or parts thereof (in this case a stretch ofsubstantially contiguous nucleotides derived from the gene of interest,or from any nucleic acid capable of encoding an orthologue, paralogue orhomologue of the protein of interest) in a sense orientation into aplant. “Sense orientation” refers to a DNA sequence that is homologousto an mRNA transcript thereof. Introduced into a plant would thereforebe at least one copy of the nucleic acid sequence. The additionalnucleic acid sequence will reduce expression of the endogenous gene,giving rise to a phenomenon known as co-suppression. The reduction ofgene expression will be more pronounced if several additional copies ofa nucleic acid sequence are introduced into the plant, as there is apositive correlation between high transcript levels and the triggeringof co-suppression.

Another example of an RNA silencing method involves the use of antisensenucleic acid sequences. An “antisense” nucleic acid sequence comprises anucleotide sequence that is complementary to a “sense” nucleic acidsequence encoding a protein, i.e. complementary to the coding strand ofa double-stranded cDNA molecule or complementary to an mRNA transcriptsequence. The antisense nucleic acid sequence is preferablycomplementary to the endogenous gene to be silenced. The complementaritymay be located in the “coding region” and/or in the “non-coding region”of a gene. The term “coding region” refers to a region of the nucleotidesequence comprising codons that are translated into amino acid residues.The term “non-coding region” refers to 5′ and 3′ sequences that flankthe coding region that are transcribed but not translated into aminoacids (also referred to as 5′ and 3′ untranslated regions).

Antisense nucleic acid sequences can be designed according to the rulesof Watson and Crick base pairing. The antisense nucleic acid sequencemay be complementary to the entire nucleic acid sequence (in this case astretch of substantially contiguous nucleotides derived from the gene ofinterest, or from any nucleic acid capable of encoding an orthologue,paralogue or homologue of the protein of interest), but may also be anoligonucleotide that is antisense to only a part of the nucleic acidsequence (including the mRNA 5′ and 3′ UTR). For example, the antisenseoligonucleotide sequence may be complementary to the region surroundingthe translation start site of an mRNA transcript encoding a polypeptide.The length of a suitable antisense oligonucleotide sequence is known inthe art and may start from about 50, 45, 40, 35, 30, 25, 20, 15 or 10nucleotides in length or less. An antisense nucleic acid sequenceaccording to the invention may be constructed using chemical synthesisand enzymatic ligation reactions using methods known in the art. Forexample, an antisense nucleic acid sequence (e.g., an antisenseoligonucleotide sequence) may be chemically synthesized using naturallyoccurring nucleotides or variously modified nucleotides designed toincrease the biological stability of the molecules or to increase thephysical stability of the duplex formed between the antisense and sensenucleic acid sequences, e.g., phosphorothioate derivatives and acridinesubstituted nucleotides may be used. Examples of modified nucleotidesthat may be used to generate the antisense nucleic acid sequences arewell known in the art. Known nucleotide modifications includemethylation, cyclization and ‘caps’ and substitution of one or more ofthe naturally occurring nucleotides with an analogue such as inosine.Other modifications of nucleotides are well known in the art.

The antisense nucleic acid sequence can be produced biologically usingan expression vector into which a nucleic acid sequence has beensubcloned in an antisense orientation (i.e., RNA transcribed from theinserted nucleic acid will be of an antisense orientation to a targetnucleic acid of interest). Preferably, production of antisense nucleicacid sequences in plants occurs by means of a stably integrated nucleicacid construct comprising a promoter, an operably linked antisenseoligonucleotide, and a terminator.

The nucleic acid molecules used for silencing in the methods of theinvention (whether introduced into a plant or generated in situ)hybridize with or bind to mRNA transcripts and/or genomic DNA encoding apolypeptide to thereby inhibit expression of the protein, e.g., byinhibiting transcription and/or translation. The hybridization can be byconventional nucleotide complementarity to form a stable duplex, or, forexample, in the case of an antisense nucleic acid sequence which bindsto DNA duplexes, through specific interactions in the major groove ofthe double helix. Antisense nucleic acid sequences may be introducedinto a plant by transformation or direct injection at a specific tissuesite. Alternatively, antisense nucleic acid sequences can be modified totarget selected cells and then administered systemically. For example,for systemic administration, antisense nucleic acid sequences can bemodified such that they specifically bind to receptors or antigensexpressed on a selected cell surface, e.g., by linking the antisensenucleic acid sequence to peptides or antibodies which bind to cellsurface receptors or antigens. The antisense nucleic acid sequences canalso be delivered to cells using the vectors described herein.

According to a further aspect, the antisense nucleic acid sequence is ana-anomeric nucleic acid sequence. An a-anomeric nucleic acid sequenceforms specific double-stranded hybrids with complementary RNA in which,contrary to the usual b-units, the strands run parallel to each other(Gaultier et al. (1987) Nucl Ac Res 15: 6625-6641). The antisensenucleic acid sequence may also comprise a 2′-o-methylribonucleotide(Inoue et al. (1987) Nucl Ac Res 15, 6131-6148) or a chimeric RNA-DNAanalogue (Inoue et al. (1987) FEBS Lett. 215, 327-330).

The reduction or substantial elimination of endogenous gene expressionmay also be performed using ribozymes. Ribozymes are catalytic RNAmolecules with ribonuclease activity that are capable of cleaving asingle-stranded nucleic acid sequence, such as an mRNA, to which theyhave a complementary region. Thus, ribozymes (e.g., hammerhead ribozymes(described in Haselhoff and Gerlach (1988) Nature 334, 585-591) can beused to catalytically cleave mRNA transcripts encoding a polypeptide,thereby substantially reducing the number of mRNA transcripts to betranslated into a polypeptide. A ribozyme having specificity for anucleic acid sequence can be designed (see for example: Cech et al. U.S.Pat. No. 4,987,071; and Cech et al. U.S. Pat. No. 5,116,742).Alternatively, mRNA transcripts corresponding to a nucleic acid sequencecan be used to select a catalytic RNA having a specific ribonucleaseactivity from a pool of RNA molecules (Bartel and Szostak (1993) Science261, 1411-1418). The use of ribozymes for gene silencing in plants isknown in the art (e.g., Atkins et al. (1994) WO 94/00012; Lenne et al.(1995) WO 95/03404; Lutziger et al. (2000) WO 00/00619; Prinsen et al.(1997) WO 97/13865 and Scott et al. (1997) WO 97/38116).

Gene silencing may also be achieved by insertion mutagenesis (forexample, T-DNA insertion or transposon insertion) or by strategies asdescribed by, among others, Angell and Baulcombe ((1999) Plant J 20(3):357-62), (Amplicon VIGS WO 98/36083), or Baulcombe (WO 99/15682).

Gene silencing may also occur if there is a mutation on an endogenousgene and/or a mutation on an isolated gene/nucleic acid subsequentlyintroduced into a plant. The reduction or substantial elimination may becaused by a non-functional polypeptide. For example, a polypeptide maybind to various interacting proteins; one or more mutation(s) and/ortruncation(s) may therefore provide for a polypeptide that is still ableto bind interacting proteins (such as receptor proteins) but that cannotexhibit its normal function (such as signalling ligand).

A further approach to gene silencing is by targeting nucleic acidsequences complementary to the regulatory region of the gene (e.g., thepromoter and/or enhancers) to form triple helical structures thatprevent transcription of the gene in target cells. See Helene, C.,Anticancer Drug Res. 6, 569-84, 1991; Helene et al., Ann. N.Y. Acad.Sci. 660, 27-36 1992; and Maher, L. J. Bioassays 14, 807-15, 1992.

Other methods, such as the use of antibodies directed to an endogenouspolypeptide for inhibiting its function in planta, or interference inthe signalling pathway in which a polypeptide is involved, will be wellknown to the skilled man. In particular, it can be envisaged thatmanmade molecules may be useful for inhibiting the biological functionof a target polypeptide, or for interfering with the signalling pathwayin which the target polypeptide is involved.

Alternatively, a screening program may be set up to identify in a plantpopulation natural variants of a gene, which variants encodepolypeptides with reduced activity. Such natural variants may also beused for example, to perform homologous recombination.

Artificial and/or natural microRNAs (miRNAs) may be used to knock outgene expression and/or mRNA translation. Endogenous miRNAs are singlestranded small RNAs of typically 19-24 nucleotides long. They functionprimarily to regulate gene expression and/or mRNA translation. Mostplant microRNAs (miRNAs) have perfect or near-perfect complementaritywith their target sequences. However, there are natural targets with upto five mismatches. They are processed from longer non-coding RNAs withcharacteristic fold-back structures by double-strand specific RNases ofthe Dicer family. Upon processing, they are incorporated in theRNA-induced silencing complex (RISC) by binding to its main component,an Argonaute protein. MiRNAs serve as the specificity components ofRISC, since they base-pair to target nucleic acids, mostly mRNAs, in thecytoplasm. Subsequent regulatory events include target mRNA cleavage anddestruction and/or translational inhibition. Effects of miRNAoverexpression are thus often reflected in decreased mRNA levels oftarget genes.

Artificial microRNAs (amiRNAs), which are typically 21 nucleotides inlength, can be genetically engineered specifically to negativelyregulate gene expression of single or multiple genes of interest.Determinants of plant microRNA target selection are well known in theart. Empirical parameters for target recognition have been defined andcan be used to aid in the design of specific amiRNAs, Schwab R, 2005.Convenient tools for design and generation of amiRNAs and theirprecursors are also available to the public, Schwab et al., 2006.

For optimal performance, the gene silencing techniques used for reducingexpression in a plant of an endogenous gene requires the use of nucleicacid sequences from monocotyledonous plants for transformation ofmonocotyledonous plants, and from dicotyledonous plants fortransformation of dicotyledonous plants. Preferably, a nucleic acidsequence from any given plant species is introduced into that samespecies. For example, a nucleic acid sequence from rice is transformedinto a rice plant. However, it is not an absolute requirement that thenucleic acid sequence to be introduced originates from the same plantspecies as the plant in which it will be introduced. It is sufficientthat there is substantial homology between the endogenous target geneand the nucleic acid to be introduced.

Described above are examples of various methods for the reduction orsubstantial elimination of expression in a plant of an endogenous gene.A person skilled in the art would readily be able to adapt theaforementioned methods for silencing so as to achieve reduction ofexpression of an endogenous gene in a whole plant or in parts thereofthrough the use of an appropriate promoter, for example.

Selectable Marker (Gene)/Reporter Gene

“Selectable marker”, “selectable marker gene” or “reporter gene”includes any gene that confers a phenotype on a cell in which it isexpressed to facilitate the identification and/or selection of cellsthat are transfected or transformed with a nucleic acid construct of theinvention. These marker genes enable the identification of a successfultransfer of the nucleic acid molecules via a series of differentprinciples. Suitable markers may be selected from markers that conferantibiotic or herbicide resistance, that introduce a new metabolic traitor that allow visual selection. Examples of selectable marker genesinclude genes conferring resistance to antibiotics (such as nptII thatphosphorylates neomycin and kanamycin, or hpt, phosphorylatinghygromycin, or genes conferring resistance to, for example, bleomycin,streptomycin, tetracyclin, chloramphenicol, ampicillin, gentamycin,geneticin (G418), spectinomycin or blasticidin), to herbicides (forexample bar which provides resistance to Basta®; aroA or gox providingresistance against glyphosate, or the genes conferring resistance to,for example, imidazolinone, phosphinothricin or sulfonylurea), or genesthat provide a metabolic trait (such as manA that allows plants to usemannose as sole carbon source or xylose isomerase for the utilisation ofxylose, or antinutritive markers such as the resistance to2-deoxyglucose). Expression of visual marker genes results in theformation of colour (for example β-glucuronidase, GUS or β-galactosidasewith its coloured substrates, for example X-Gal), luminescence (such asthe luciferin/luceferase system) or fluorescence (Green FluorescentProtein, GFP, and derivatives thereof). This list represents only asmall number of possible markers. The skilled worker is familiar withsuch markers. Different markers are preferred, depending on the organismand the selection method.

It is known that upon stable or transient integration of nucleic acidsinto plant cells, only a minority of the cells takes up the foreign DNAand, if desired, integrates it into its genome, depending on theexpression vector used and the transfection technique used. To identifyand select these integrants, a gene coding for a selectable marker (suchas the ones described above) is usually introduced into the host cellstogether with the gene of interest. These markers can for example beused in mutants in which these genes are not functional by, for example,deletion by conventional methods. Furthermore, nucleic acid moleculesencoding a selectable marker can be introduced into a host cell on thesame vector that comprises the sequence encoding the polypeptides of theinvention or used in the methods of the invention, or else in a separatevector. Cells which have been stably transfected with the introducednucleic acid can be identified for example by selection (for example,cells which have integrated the selectable marker survive whereas theother cells die).

Since the marker genes, particularly genes for resistance to antibioticsand herbicides, are no longer required or are undesired in thetransgenic host cell once the nucleic acids have been introducedsuccessfully, the process according to the invention for introducing thenucleic acids advantageously employs techniques which enable the removalor excision of these marker genes. One such a method is what is known asco-transformation. The co-transformation method employs two vectorssimultaneously for the transformation, one vector bearing the nucleicacid according to the invention and a second bearing the marker gene(s).A large proportion of transformants receives or, in the case of plants,comprises (up to 40% or more of the transformants), both vectors. Incase of transformation with Agrobacteria, the transformants usuallyreceive only a part of the vector, i.e. the sequence flanked by theT-DNA, which usually represents the expression cassette. The markergenes can subsequently be removed from the transformed plant byperforming crosses. In another method, marker genes integrated into atransposon are used for the transformation together with desired nucleicacid (known as the Ac/Ds technology). The transformants can be crossedwith a transposase source or the transformants are transformed with anucleic acid construct conferring expression of a transposase,transiently or stable. In some cases (approx. 10%), the transposon jumpsout of the genome of the host cell once transformation has taken placesuccessfully and is lost. In a further number of cases, the transposonjumps to a different location. In these cases the marker gene must beeliminated by performing crosses. In microbiology, techniques weredeveloped which make possible, or facilitate, the detection of suchevents. A further advantageous method relies on what is known asrecombination systems; whose advantage is that elimination by crossingcan be dispensed with. The best-known system of this type is what isknown as the Cre/lox system. Cre1 is a recombinase that removes thesequences located between the loxP sequences. If the marker gene isintegrated between the loxP sequences, it is removed once transformationhas taken place successfully, by expression of the recombinase. Furtherrecombination systems are the HIN/HIX, FLP/FRT and REP/STB system(Tribble et al., J. Biol. Chem., 275, 2000: 22255-22267; Velmurugan etal., J. Cell Biol., 149, 2000: 553-566). A site-specific integrationinto the plant genome of the nucleic acid sequences according to theinvention is possible. Naturally, these methods can also be applied tomicroorganisms such as yeast, fungi or bacteria.

Transgenic/Transgene/Recombinant

For the purposes of the invention, “transgenic”, “transgene” or“recombinant” means with regard to, for example, a nucleic acidsequence, an expression cassette, gene construct or a vector comprisingthe nucleic acid sequence or an organism transformed with the nucleicacid sequences, expression cassettes or vectors according to theinvention, all those constructions brought about by recombinant methodsin which either

-   -   (a) the nucleic acid sequences encoding proteins useful in the        methods of the invention, or    -   (b) genetic control sequence(s) which is operably linked with        the nucleic acid sequence according to the invention, for        example a promoter, or    -   (c) a) and b)        are not located in their natural genetic environment or have        been modified by recombinant methods, it being possible for the        modification to take the form of, for example, a substitution,        addition, deletion, inversion or insertion of one or more        nucleotide residues. The natural genetic environment is        understood as meaning the natural genomic or chromosomal locus        in the original plant or the presence in a genomic library. In        the case of a genomic library, the natural genetic environment        of the nucleic acid sequence is preferably retained, at least in        part. The environment flanks the nucleic acid sequence at least        on one side and has a sequence length of at least 50 bp,        preferably at least 500 bp, especially preferably at least 1000        bp, most preferably at least 5000 bp. A naturally occurring        expression cassette—for example the naturally occurring        combination of the natural promoter of the nucleic acid        sequences with the corresponding nucleic acid sequence encoding        a polypeptide useful in the methods of the present invention, as        defined above—becomes a transgenic expression cassette when this        expression cassette is modified by non-natural, synthetic        (“artificial”) methods such as, for example, mutagenic        treatment. Suitable methods are described, for example, in U.S.        Pat. No. 5,565,350 or WO 00/15815.

A transgenic plant for the purposes of the invention is thus understoodas meaning, as above, that the nucleic acids used in the method of theinvention are not at their natural locus in the genome of said plant, itbeing possible for the nucleic acids to be expressed homologously orheterologously. However, as mentioned, transgenic also means that, whilethe nucleic acids according to the invention or used in the inventivemethod are at their natural position in the genome of a plant, thesequence has been modified with regard to the natural sequence, and/orthat the regulatory sequences of the natural sequences have beenmodified. Transgenic is preferably understood as meaning the expressionof the nucleic acids according to the invention at an unnatural locus inthe genome, i.e. homologous or, preferably, heterologous expression ofthe nucleic acids takes place. Preferred transgenic plants are mentionedherein.

Transformation

The term “introduction” or “transformation” as referred to hereinencompasses the transfer of an exogenous polynucleotide into a hostcell, irrespective of the method used for transfer. Plant tissue capableof subsequent clonal propagation, whether by organogenesis orembryogenesis, may be transformed with a genetic construct of thepresent invention and a whole plant regenerated there from. Theparticular tissue chosen will vary depending on the clonal propagationsystems available for, and best suited to, the particular species beingtransformed. Exemplary tissue targets include leaf disks, pollen,embryos, cotyledons, hypocotyls, megagametophytes, callus tissue,existing meristematic tissue (e.g., apical meristem, axillary buds, androot meristems), and induced meristem tissue (e.g., cotyledon meristemand hypocotyl meristem). The polynucleotide may be transiently or stablyintroduced into a host cell and may be maintained non-integrated, forexample, as a plasmid. Alternatively, it may be integrated into the hostgenome. The resulting transformed plant cell may then be used toregenerate a transformed plant in a manner known to persons skilled inthe art.

The transfer of foreign genes into the genome of a plant is calledtransformation. Transformation of plant species is now a fairly routinetechnique. Advantageously, any of several transformation methods may beused to introduce the gene of interest into a suitable ancestor cell.The methods described for the transformation and regeneration of plantsfrom plant tissues or plant cells may be utilized for transient or forstable transformation. Transformation methods include the use ofliposomes, electroporation, chemicals that increase free DNA uptake,injection of the DNA directly into the plant, particle gun bombardment,transformation using viruses or pollen and microprojection. Methods maybe selected from the calcium/polyethylene glycol method for protoplasts(Krens, F. A. et al., (1982) Nature 296, 72-74; Negrutiu I et al. (1987)Plant Mol Biol 8: 363-373); electroporation of protoplasts (Shillito R.D. et al. (1985) Bio/Technol 3, 1099-1102); microinjection into plantmaterial (Crossway A et al., (1986) Mol. Gen Genet 202: 179-185); DNA orRNA-coated particle bombardment (Klein T M et al., (1987) Nature 327:70) infection with (non-integrative) viruses and the like. Transgenicplants, including transgenic crop plants, are preferably produced viaAgrobacterium-mediated transformation. An advantageous transformationmethod is the transformation in planta. To this end, it is possible, forexample, to allow the agrobacteria to act on plant seeds or to inoculatethe plant meristem with agrobacteria. It has proved particularlyexpedient in accordance with the invention to allow a suspension oftransformed agrobacteria to act on the intact plant or at least on theflower primordia. The plant is subsequently grown on until the seeds ofthe treated plant are obtained (Clough and Bent, Plant J. (1998) 16,735-743). Methods for Agrobacterium-mediated transformation of riceinclude well known methods for rice transformation, such as thosedescribed in any of the following: European patent application EP1198985 A1, Aldemita and Hodges (Planta 199: 612-617, 1996); Chan et al.(Plant Mol Biol 22 (3): 491-506, 1993), Hiei et al. (Plant J 6 (2):271-282, 1994), which disclosures are incorporated by reference hereinas if fully set forth. In the case of corn transformation, the preferredmethod is as described in either Ishida et al. (Nat. Biotechnol 14(6):745-50, 1996) or Frame et al. (Plant Physiol 129(1): 13-22, 2002), whichdisclosures are incorporated by reference herein as if fully set forth.Said methods are further described by way of example in B. Jenes et al.,Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineeringand Utilization, eds. S. D. Kung and R. Wu, Academic Press (1993)128-143 and in Potrykus Annu. Rev. Plant Physiol. Plant Molec. Biol. 42(1991) 205-225). The nucleic acids or the construct to be expressed ispreferably cloned into a vector, which is suitable for transformingAgrobacterium tumefaciens, for example pBin19 (Bevan et al., Nucl. AcidsRes. 12 (1984) 8711). Agrobacteria transformed by such a vector can thenbe used in known manner for the transformation of plants, such as plantsused as a model, like Arabidopsis (Arabidopsis thaliana is within thescope of the present invention not considered as a crop plant), or cropplants such as, by way of example, tobacco plants, for example byimmersing bruised leaves or chopped leaves in an agrobacterial solutionand then culturing them in suitable media. The transformation of plantsby means of Agrobacterium tumefaciens is described, for example, byHöfgen and Willmitzer in Nucl. Acid Res. (1988) 16, 9877 or is knowninter alia from F.F. White, Vectors for Gene Transfer in Higher Plants;in Transgenic Plants, Vol. 1, Engineering and Utilization, eds. S. D.Kung and R. Wu, Academic Press, 1993, pp. 15-38.

In addition to the transformation of somatic cells, which then have tobe regenerated into intact plants, it is also possible to transform thecells of plant meristems and in particular those cells which developinto gametes. In this case, the transformed gametes follow the naturalplant development, giving rise to transgenic plants. Thus, for example,seeds of Arabidopsis are treated with agrobacteria and seeds areobtained from the developing plants of which a certain proportion istransformed and thus transgenic [Feldman, K A and Marks M D (1987). MolGen Genet 208:274-289; Feldmann K (1992). In: C Koncz, N-H Chua and JShell, eds, Methods in Arabidopsis Research. Word Scientific, Singapore,pp. 274-289]. Alternative methods are based on the repeated removal ofthe inflorescences and incubation of the excision site in the center ofthe rosette with transformed agrobacteria, whereby transformed seeds canlikewise be obtained at a later point in time (Chang (1994). Plant J. 5:551-558; Katavic (1994). Mol Gen Genet, 245: 363-370). However, anespecially effective method is the vacuum infiltration method with itsmodifications such as the “floral dip” method. In the case of vacuuminfiltration of Arabidopsis, intact plants under reduced pressure aretreated with an agrobacterial suspension [Bechthold, N (1993). C R AcadSci Paris Life Sci, 316: 1194-1199], while in the case of the “floraldip” method the developing floral tissue is incubated briefly with asurfactant-treated agrobacterial suspension [Clough, S J and Bent, A F(1998). The Plant J. 16, 735-743]. A certain proportion of transgenicseeds are harvested in both cases, and these seeds can be distinguishedfrom non-transgenic seeds by growing under the above-described selectiveconditions. In addition the stable transformation of plastids is ofadvantages because plastids are inherited maternally is most cropsreducing or eliminating the risk of transgene flow through pollen. Thetransformation of the chloroplast genome is generally achieved by aprocess which has been schematically displayed in Klaus et al., 2004[Nature Biotechnology 22 (2), 225-229]. Briefly the sequences to betransformed are cloned together with a selectable marker gene betweenflanking sequences homologous to the chloroplast genome. Thesehomologous flanking sequences direct site specific integration into theplastome. Plastidal transformation has been described for many differentplant species and an overview is given in Bock (2001) Transgenicplastids in basic research and plant biotechnology. J Mol Biol. 2001Sep. 21; 312 (3):425-38 or Maliga, P (2003) Progress towardscommercialization of plastid transformation technology. TrendsBiotechnol. 21, 20-28. Further biotechnological progress has recentlybeen reported in form of marker free plastid transformants, which can beproduced by a transient co-integrated maker gene (Klaus et al., 2004,Nature Biotechnology 22(2), 225-229).

T-DNA Activation Tagging

T-DNA activation tagging (Hayashi et al. Science (1992) 1350-1353),involves insertion of T-DNA, usually containing a promoter (may also bea translation enhancer or an intron), in the genomic region of the geneof interest or 10 kb up- or downstream of the coding region of a gene ina configuration such that the promoter directs expression of thetargeted gene. Typically, regulation of expression of the targeted geneby its natural promoter is disrupted and the gene falls under thecontrol of the newly introduced promoter. The promoter is typicallyembedded in a T-DNA. This T-DNA is randomly inserted into the plantgenome, for example, through Agrobacterium infection and leads tomodified expression of genes near the inserted T-DNA. The resultingtransgenic plants show dominant phenotypes due to modified expression ofgenes close to the introduced promoter.

Tilling

TILLING (Targeted Induced Local Lesions In Genomes) is a mutagenesistechnology useful to generate and/or identify nucleic acids encodingproteins with modified expression and/or activity. TILLING also allowsselection of plants carrying such mutant variants. These mutant variantsmay exhibit modified expression, either in strength or in location or intiming (if the mutations affect the promoter for example). These mutantvariants may exhibit higher activity than that exhibited by the gene inits natural form. TILLING combines high-density mutagenesis withhigh-throughput screening methods. The steps typically followed inTILLING are: (a) EMS mutagenesis (Redei G P and Koncz C (1992) InMethods in Arabidopsis Research, Koncz C, Chua N H, Schell J, eds.Singapore, World Scientific Publishing Co, pp. 16-82; Feldmann et al.,(1994) In Meyerowitz E M, Somerville C R, eds, Arabidopsis. Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., pp 137-172; LightnerJ and Caspar T (1998) In J Martinez-Zapater, J Salinas, eds, Methods onMolecular Biology, Vol. 82. Humana Press, Totowa, N.J., pp 91-104); (b)DNA preparation and pooling of individuals; (c) PCR amplification of aregion of interest; (d) denaturation and annealing to allow formation ofheteroduplexes; (e) DHPLC, where the presence of a heteroduplex in apool is detected as an extra peak in the chromatogram; (f)identification of the mutant individual; and (g) sequencing of themutant PCR product. Methods for TILLING are well known in the art(McCallum et al., (2000) Nat Biotechnol 18: 455-457; reviewed by Stemple(2004) Nat Rev Genet 5(2): 145-50).

Homologous Recombination

Homologous recombination allows introduction in a genome of a selectednucleic acid at a defined selected position. Homologous recombination isa standard technology used routinely in biological sciences for lowerorganisms such as yeast or the moss Physcomitrella. Methods forperforming homologous recombination in plants have been described notonly for model plants (Offring a et al. (1990) EMBO J 9(10): 3077-84)but also for crop plants, for example rice (Terada et al. (2002) NatBiotech 20(10): 1030-4; Iida and Terada (2004) Curr Opin Biotech 15(2):132-8).

Yield

The term “yield” in general means a measurable produce of economicvalue, typically related to a specified crop, to an area, and to aperiod of time. Individual plant parts directly contribute to yieldbased on their number, size and/or weight, or the actual yield is theyield per acre for a crop and year, which is determined by dividingtotal production (includes both harvested and appraised production) byplanted acres.

Early Vigour

Early vigour (active healthy well-balanced growth especially duringearly stages of plant growth) may result from increased plant fitnessdue to, for example, the plants being better adapted to theirenvironment (i.e. optimizing the use of energy resources andpartitioning between shoot and root). Plants having early vigour alsoshow increased seedling survival and a better establishment of the crop,which often results in highly uniform fields (with the crop growing inuniform manner, i.e. with the majority of plants reaching the variousstages of development at substantially the same time), and often betterand higher yield. Therefore, early vigour may be determined by measuringvarious factors, such as thousand kernel weight, percentage germination,percentage emergence, seedling growth, seedling height, root length,root and shoot biomass and many more.

Increase/Improve/Enhance

The terms “increase”, “improve” or “enhance” are interchangeable andshall mean in the sense of the application at least a 5%, 6%, 7%, 8%, 9%or 10%, preferably at least 15% or 20%, more preferably 25%, 30%, 35% or40% more yield and/or growth in comparison to control plants as definedherein.

Seed Yield

Increased seed yield may manifest itself as one or more of thefollowing: a) an increase in seed biomass (total seed weight) which maybe on an individual seed basis and/or per plant and/or per hectare oracre; b) increased number of flowers per plant; c) increased number of(filled) seeds; d) increased seed filling rate (which is expressed asthe ratio between the number of filled seeds divided by the total numberof seeds); e) increased harvest index, which is expressed as a ratio ofthe yield of harvestable parts, such as seeds, divided by the totalbiomass; and f) increased thousand kernel weight (TKW), which isextrapolated from the number of filled seeds counted and their totalweight. An increased TKW may result from an increased seed size and/orseed weight, and may also result from an increase in embryo and/orendosperm size.

An increase in seed yield may also be manifested as an increase in seedsize and/or seed volume. Furthermore, an increase in seed yield may alsomanifest itself as an increase in seed area and/or seed length and/orseed width and/or seed perimeter. Increased yield may also result inmodified architecture, or may occur because of modified architecture.

Plant

The term “plant” as used herein encompasses whole plants, ancestors andprogeny of the plants and plant parts, including seeds, shoots, stems,leaves, roots (including tubers), flowers, and tissues and organs,wherein each of the aforementioned comprise the gene/nucleic acid ofinterest. The term “plant” also encompasses plant cells, suspensioncultures, callus tissue, embryos, meristematic regions, gametophytes,sporophytes, pollen and microspores, again wherein each of theaforementioned comprises the gene/nucleic acid of interest.

Plants that are particularly useful in the methods of the inventioninclude all plants which belong to the superfamily Viridiplantae, inparticular monocotyledonous and dicotyledonous plants including fodderor forage legumes, ornamental plants, food crops, trees or shrubsselected from the list comprising Acer spp., Actinidia spp., Abelmoschusspp., Agave sisalana, Agropyron spp., Agrostis stolonifera, Allium spp.,Amaranthus spp., Ammophila arenaria, Ananas comosus, Annona spp., Apiumgraveolens, Arachis spp, Artocarpus spp., Asparagus officinalis, Avenaspp. (e.g. Avena sativa, Avena fatua, Avena byzantina, Avena fatua var.sativa, Avena hybrida), Averrhoa carambola, Bambusa sp., Benincasahispida, Bertholletia excelsea, Beta vulgaris, Brassica spp. (e.g.Brassica napus, Brassica rapa ssp. [canola, oilseed rape, turnip rape]),Cadaba farinosa, Camellia sinensis, Canna indica, Cannabis sativa,Capsicum spp., Carex elata, Carica papaya, Carissa macrocarpa, Caryaspp., Carthamus tinctorius, Castanea spp., Ceiba pentandra, Cichoriumendivia, Cinnamomum spp., Citrullus lanatus, Citrus spp., Cocos spp.,Coffea spp., Colocasia esculenta, Cola spp., Corchorus sp., Coriandrumsativum, Corylus spp., Crataegus spp., Crocus sativus, Cucurbita spp.,Cucumis spp., Cynara spp., Daucus carota, Desmodium spp., Dimocarpuslongan, Dioscorea spp., Diospyros spp., Echinochloa spp., Elaeis (e.g.Elaeis guineensis, Elaeis oleifera), Eleusine coracana, Erianthus sp.,Eriobotrya japonica, Eucalyptus sp., Eugenia uniflora, Fagopyrum spp.,Fagus spp., Festuca arundinacea, Ficus carica, Fortunella spp., Fragariaspp., Ginkgo biloba, Glycine spp. (e.g. Glycine max, Soja hispida orSoja max), Gossypium hirsutum, Helianthus spp. (e.g. Helianthus annuus),Hemerocallis fulva, Hibiscus spp., Hordeum spp. (e.g. Hordeum vulgare),Ipomoea batatas, Juglans spp., Lactuca sativa, Lathyrus spp., Lensculinaris, Linum usitatissimum, Litchi chinensis, Lotus spp., Luffaacutangula, Lupinus spp., Luzula sylvatica, Lycopersicon spp. (e.g.Lycopersicon esculentum, Lycopersicon lycopersicum, Lycopersiconpyriforme), Macrotyloma spp., Malus spp., Malpighia emarginata, Mammeaamericana, Mangifera indica, Manihot spp., Manilkara zapota, Medicagosativa, Melilotus spp., Mentha spp., Miscanthus sinensis, Momordicaspp., Morus nigra, Musa spp., Nicotiana spp., Olea spp., Opuntia spp.,Ornithopus spp., Oryza spp. (e.g. Oryza sativa, Oryza latifolia),Panicum miliaceum, Panicum virgatum, Passiflora edulis, Pastinacasativa, Pennisetum sp., Persea spp., Petroselinum crispum, Phalarisarundinacea, Phaseolus spp., Phleum pratense, Phoenix spp., Phragmitesaustralis, Physalis spp., Pinus spp., Pistacia vera, Pisum spp., Poaspp., Populus spp., Prosopis spp., Prunus spp., Psidium spp., Punicagranatum, Pyrus communis, Quercus spp., Raphanus sativus, Rheumrhabarbarum, Ribes spp., Ricinus communis, Rubus spp., Saccharum spp.,Salix sp., Sambucus spp., Secale cereale, Sesamum spp., Sinapis sp.,Solanum spp. (e.g. Solanum tuberosum, Solanum integrifolium or Solanumlycopersicum), Sorghum bicolor, Spinacia spp., Syzygium spp., Tagetesspp., Tamarindus indica, Theobroma cacao, Trifolium spp., Triticosecalerimpaui, Triticum spp. (e.g. Triticum aestivum, Triticum durum, Triticumturgidum, Triticum hybernum, Triticum macha, Triticum sativum orTriticum vulgare), Tropaeolum minus, Tropaeolum majus, Vaccinium spp.,Vicia spp., Vigna spp., Viola odorata, Vitis spp., Zea mays, Zizaniapalustris, Ziziphus spp., amongst others.

DETAILED DESCRIPTION OF THE INVENTION Detailed Description for HAL3

According to a first embodiment of the present invention, there isprovided a method for increasing plant yield relative to control plants,comprising preferentially increasing expression of a nucleic acidencoding a HAL3 polypeptide in expanding tissues of a plant.

Any reference hereinafter to a “protein useful in the methods of theinvention” is taken to mean a HAL3 polypeptide as defined herein. Anyreference hereinafter to a “nucleic acid useful in the methods of theinvention” is taken to mean a nucleic acid capable of encoding such anHAL3 polypeptide. The nucleic acid to be introduced into a plant (andtherefore useful in performing the methods of the invention) is anynucleic acid encoding the type of protein which will now be described,hereafter also named “HAL3 nucleic acid” or “HAL3 gene”.

A “reference”, “reference plant”, “control”, “control plant”, “wildtype” or “wild type plant” is in particular a cell, a tissue, an organ,a plant, or a part thereof, which was not produced according to themethod of the invention. Accordingly, the terms “wild type”, “control”or “reference” are exchangeable and can be a cell or a part of the plantsuch as an organelle or tissue, or a plant, which was not modified ortreated according to the herein described method according to theinvention. Accordingly, the cell or a part of the plant such as anorganelle or a plant used as wild type, control or reference correspondsto the cell, plant or part thereof as much as possible and is in anyother property but in the result of the process of the invention asidentical to the subject matter of the invention as possible. Thus, thewild type, control or reference is treated identically or as identicalas possible, saying that only conditions or properties might bedifferent which do not influence the quality of the tested property.That means in other words that the wild type denotes (1) a plant, whichcarries the unaltered or not modulated form of a gene or allele or (2)the starting material/plant from which the plants produced by theprocess or method of the invention are derived.

Preferably, any comparison between the wild type plants and the plantsproduced by the method of the invention is carried out under analogousconditions. The term “analogous conditions” means that all conditionssuch as, for example, culture or growing conditions, assay conditions(such as buffer composition, temperature, substrates, pathogen strain,concentrations and the like) are kept identical between the experimentsto be compared.

The “reference”, “control”, or “wild type” is preferably a subject, e.g.an organelle, a cell, a tissue, a plant, which was not modulated,modified or treated according to the herein described process of theinvention and is in any other property as similar to the subject matterof the invention as possible. The reference, control or wild type is inits genome, transcriptome, proteome or metabolome as similar as possibleto the subject of the present invention. Preferably, the term“reference-” “control-” or “wild type-”-organelle, -cell, -tissue orplant, relates to an organelle, cell, tissue or plant, which is nearlygenetically identical to the organelle, cell, tissue or plant, of thepresent invention or a part thereof preferably 95%, more preferred are98%, even more preferred are 99.00%, in particular 99.10%, 99.30%,99.50%, 99.70%, 99.90%, 99.99%, 99, 999% or more. Most preferable the“reference”, “control”, or “wild type” is preferably a subject, e.g. anorganelle, a cell, a tissue, a plant, which is genetically identical tothe plant, cell organelle used according to the method of the inventionexcept that nucleic acid molecules or the gene product encoded by themare changed, modulated or modified according to the inventive method.

In case, a control, reference or wild type differing from the subject ofthe present invention only by not being subject of the method of theinvention can not be provided, a control, reference or wild type can bea plant in which the cause for the modulation of the activity conferringthe increase of the metabolites as described under examples.

The increase referred to the activity of the polypeptide amounts in acell, a tissue, a organelle, an organ or an organism or a part thereofpreferably to at least 5%, preferably to at least 10% or at to least15%, especially preferably to at least 20%, 25%, 30% or more, veryespecially preferably are to at least 40%, 50% or 60%, most preferablyare to at least 70% or more in comparison to the control, reference orwild type.

The term “increased yield” is taken to mean an increase in biomass(weight) of one or more parts of a plant, which may include aboveground(harvestable) parts and/or (harvestable) parts below ground. Preferably,the increase in yield is at least 10% over the yield of correspondingwild type plants.

In particular, such harvestable parts are seeds, and performance of themethods of the invention results in plants having increased seed yieldrelative to the seed yield of control plants.

The term “expression” or “gene expression” means the appearance of aphenotypic trait as a consequence of the transcription of a specificgene or specific genes. The term “expression” or “gene expression” inparticular means the transcription of a gene or genes into structuralRNA (rRNA, tRNA) or mRNA with subsequent translation of the latter intoa protein. The process includes transcription of DNA, processing of theresulting mRNA product and its translation into an active protein.

Performance of the methods of the invention gives plants having enhancedyield-related traits. In particular performance of the methods of theinvention gives plants having increased yield, especially increased seedyield relative to control plants. The terms “yield” and “seed yield” aredescribed in more detail in the “definitions” section herein.

Reference herein to enhanced yield-related traits is taken to mean anincrease in biomass (weight) of one or more parts of a plant, which mayinclude aboveground (harvestable) parts and/or (harvestable) parts belowground. In particular, such harvestable parts are seeds, and performanceof the methods of the invention results in plants having increased seedyield relative to the seed yield of suitable control plants.

Taking corn as an example, a yield increase may be manifested as one ormore of the following: increase in the number of plants per hectare oracre, an increase in the number of ears per plant, an increase in thenumber of rows, number of kernels per row, kernel weight, thousandkernel weight, ear length/diameter, increase in the seed filling rate(which is the number of filled seeds divided by the total number ofseeds and multiplied by 100), among others. Taking rice as an example, ayield increase may manifest itself as an increase in one or more of thefollowing: number of plants per hectare or acre, number of panicles perplant, number of spikelets per panicle, number of flowers (florets) perpanicle (which is expressed as a ratio of the number of filled seedsover the number of primary panicles), increase in the seed filling rate(which is the number of filled seeds divided by the total number ofseeds and multiplied by 100), increase in thousand kernel weight, amongothers.

According to a preferred feature of the present invention, performanceof the methods of the invention gives plants having increased seed yieldrelative to control plants. Therefore according to the presentinvention, there is provided a method for increasing seed yield inplants relative to the seed yield of control plants, the methodcomprising preferentially increasing expression of a nucleic acidencoding a HAL3 polypeptide in shoot tissues, preferably in expandingtissues of a plant shoot.

Since the transgenic plants according to the present invention haveincreased yield, it is likely that these plants exhibit an increasedgrowth rate (during at least part of their life cycle), relative to thegrowth rate of control plants at a corresponding stage in their lifecycle.

The increased growth rate may be specific to one or more parts of aplant (including seeds), or may be throughout substantially the wholeplant. Plants having an increased growth rate may have a shorter lifecycle. The life cycle of a plant may be taken to mean the time needed togrow from a dry mature seed up to the stage where the plant has produceddry mature seeds, similar to the starting material. This life cycle maybe influenced by factors such as early vigour, growth rate, greennessindex, flowering time and speed of seed maturation. The increase ingrowth rate may take place at one or more stages in the life cycle of aplant or during substantially the whole plant life cycle. Increasedgrowth rate during the early stages in the life cycle of a plant mayreflect enhanced vigour. The increase in growth rate may alter theharvest cycle of a plant allowing plants to be sown later and/orharvested sooner than would otherwise be possible (a similar effect maybe obtained with earlier flowering time). If the growth rate issufficiently increased, it may allow for the further sowing of seeds ofthe same plant species (for example sowing and harvesting of rice plantsfollowed by sowing and harvesting of further rice plants all within oneconventional growing period). Similarly, if the growth rate issufficiently increased, it may allow for the further sowing of seeds ofdifferent plants species (for example the sowing and harvesting of cornplants followed by, for example, the sowing and optional harvesting ofsoybean, potato or any other suitable plant). Harvesting additionaltimes from the same rootstock in the case of some crop plants may alsobe possible. Altering the harvest cycle of a plant may lead to anincrease in annual biomass production per acre (due to an increase inthe number of times (say in a year) that any particular plant may begrown and harvested). An increase in growth rate may also allow for thecultivation of transgenic plants in a wider geographical area than theirwild-type counterparts, since the territorial limitations for growing acrop are often determined by adverse environmental conditions either atthe time of planting (early season) or at the time of harvesting (lateseason). Such adverse conditions may be avoided if the harvest cycle isshortened. The growth rate may be determined by deriving variousparameters from growth curves, such parameters may be: T-Mid (the timetaken for plants to reach 50% of their maximal size) and T-90 (timetaken for plants to reach 90% of their maximal size), amongst others.

According to a preferred feature of the present invention, performanceof the methods of the invention gives plants having an increased growthrate relative to control plants. Therefore, according to the presentinvention, there is provided a method for increasing the growth rate ofplants, which method comprises modulating expression, preferablyincreasing expression, in a plant of a nucleic acid encoding a HAL3polypeptide as defined herein.

An increase in yield and/or growth rate occurs whether the plant isunder non-stress conditions or whether the plant is exposed to variousstresses compared to control plants. Plants typically respond toexposure to stress by growing more slowly. In conditions of severestress, the plant may even stop growing altogether. Mild stress on theother hand is defined herein as being any stress to which a plant isexposed which does not result in the plant ceasing to grow altogetherwithout the capacity to resume growth. Mild stress in the sense of theinvention leads to a reduction in the growth of the stressed plants ofless than 40%, 35% or 30%, preferably less than 25%, 20% or 15%, morepreferably less than 14%, 13%, 12%, 11% or 10% or less in comparison tothe control plant under non-stress conditions. Due to advances inagricultural practices (irrigation, fertilization, pesticide treatments)severe stresses are not often encountered in cultivated crop plants. Asa consequence, the compromised growth induced by mild stress is often anundesirable feature for agriculture. Mild stresses are the everydaybiotic and/or abiotic (environmental) stresses to which a plant isexposed. Abiotic stresses may be due to drought or excess water,anaerobic stress, salt stress, chemical toxicity, oxidative stress andhot, cold or freezing temperatures. The abiotic stress may be an osmoticstress caused by a water stress (particularly due to drought), saltstress, oxidative stress or an ionic stress. Biotic stresses aretypically those stresses caused by pathogens, such as bacteria, viruses,fungi and insects.

In particular, the methods of the present invention may be performedunder non-stress conditions or under conditions of mild drought to giveplants having increased yield relative to control plants. As reported inWang et al. (Planta (2003) 218: 1-14), abiotic stress leads to a seriesof morphological, physiological, biochemical and molecular changes thatadversely affect plant growth and productivity. Drought, salinity,extreme temperatures and oxidative stress are known to be interconnectedand may induce growth and cellular damage through similar mechanisms.Rabbani et al. (Plant Physiol (2003) 133: 1755-1767) describes aparticularly high degree of “cross talk” between drought stress andhigh-salinity stress. For example, drought and/or salinisation aremanifested primarily as osmotic stress, resulting in the disruption ofhomeostasis and ion distribution in the cell. Oxidative stress, whichfrequently accompanies high or low temperature, salinity or droughtstress, may cause denaturing of functional and structural proteins. As aconsequence, these diverse environmental stresses often activate similarcell signaling pathways and cellular responses, such as the productionof stress proteins, up-regulation of anti-oxidants, accumulation ofcompatible solutes and growth arrest. The term “non-stress” conditionsas used herein are those environmental conditions that allow optimalgrowth of plants. Persons skilled in the art are aware of normal soilconditions and climatic conditions for a given location.

Performance of the methods of the invention gives plants grown undernon-stress conditions or under mild drought conditions increased yieldrelative to suitable control plants grown under comparable conditions.Therefore, according to the present invention, there is provided amethod for increasing yield in plants grown under non-stress conditionsor under mild drought conditions, which method comprises increasingexpression in a plant of a nucleic acid encoding a HAL3 polypeptide.

Performance of the methods of the invention gives plants grown underconditions of nutrient deficiency, particularly under conditions ofnitrogen deficiency, increased yield relative to control plants grownunder comparable conditions. Therefore, according to the presentinvention, there is provided a method for increasing yield in plantsgrown under conditions of nutrient deficiency, which method comprisesincreasing expression in a plant of a nucleic acid encoding a HAL3polypeptide. Nutrient deficiency may result from a lack or excess ofnutrients such as nitrogen, phosphates and other phosphorous-containingcompounds, potassium, calcium, cadmium, magnesium, manganese, iron andboron, amongst others.

According to a second preferred feature of the invention, performance ofthe methods of the invention gives plants having increased plant vigourrelative to control plants, particularly during the early stages ofplant development (typically three, four weeks post germination in thecase of rice and maize, but this will vary from species to species)leading to early vigour. Therefore, according to the present invention,there is provided a method for increasing the plant early vigour, whichmethod comprises modulating, preferably increasing expression in a plantof a nucleic acid encoding a HAL3 polypeptide. Preferably the increasein seedling vigour is achieved by expressing the nucleic acid encodingthe HAL3 polypeptide under the control of a shoot specific promoter.There is also provided a method for producing plants having early vigourrelative to control plants, which method comprises modulating,preferably increasing, expression in a plant of a nucleic acid encodinga HAL3 polypeptide.

Early vigour may also result from increased plant fitness due to, forexample, the plants being better adapted to their environment (i.e.optimizing the use of energy resources and partitioning between shootand root). Plants having early vigour also show increased seedlingsurvival and a better establishment of the crop, which often results inhighly uniform fields (with the crop growing in uniform manner, i.e.with the majority of plants reaching the various stages of developmentat substantially the same time), and often better and higher yield.Therefore, early vigour may be determined by measuring various factors,such as thousand kernel weight, percentage germination, percentageemergence, seedling growth, seedling height, root length, root and shootbiomass and many more.

The methods of the invention are advantageously applicable to any plant.

Plants that are particularly useful in the methods of the inventioninclude all plants which belong to the superfamily Viridiplantae, inparticular monocotyledonous and dicotyledonous plants including fodderor forage legumes, ornamental plants, food crops, trees or shrubs.According to a preferred embodiment of the present invention, the plantis a crop plant. Examples of crop plants include soybean, sunflower,canola, alfalfa, rapeseed, cotton, tomato, potato and tobacco. Furtherpreferably, the plant is a monocotyledonous plant. Examples ofmonocotyledonous plants include sugarcane. More preferably the plant isa cereal. Examples of cereals include rice, maize, wheat, barley,millet, rye, triticale, sorghum and oats.

Other advantageous plants are selected from the group consisting ofAsteraceae such as the genera Helianthus, Tagetes e.g. the speciesHelianthus annus [sunflower], Tagetes lucida, Tagetes erecta or Tagetestenuifolia [Marigold], Brassicaceae such as the genera Brassica,Arabadopsis e.g. the species Brassica napus, Brassica rapa ssp. [canola,oilseed rape, turnip rape] or Arabidopsis thaliana. Fabaceae such as thegenera Glycine e.g. the species Glycine max, Soja hispida or Soja max[soybean]. Linaceae such as the genera Linum e.g. the species Linumusitatissimum, [flax, linseed]; Poaceae such as the genera Hordeum,Secale, Avena, Sorghum, Oryza, Zea, Triticum e.g. the species Hordeumvulgare [barley]; Secale cereale [rye], Avena sativa, Avena fatua, Avenabyzantina, Avena fatua var. sativa, Avena hybrida [oat], Sorghum bicolor[Sorghum, millet], Oryza sativa, Oryza latifolia [rice], Zea mays [corn,maize] Triticum aestivum, Triticum durum, Triticum turgidum, Triticumhybernum, Triticum macha, Triticum sativum or Triticum vulgare [wheat,bread wheat, common wheat]; Solanaceae such as the genera Solanum,Lycopersicon e.g. the species Solanum tuberosum [potato], Lycopersiconesculentum, Lycopersicon lycopersicum, Lycopersicon pyriforme, Solanumintegrifolium or Solanum lycopersicum [tomato].

The term “HAL3 polypeptide” as defined herein refers to a flavoproteinbelonging to the superfamily HFCD (Homo-oligomeric Flavin containing CysDecarboxylases; Kupke J. Biol. Chem. 276, 27597-27604, 2001). Theseproteins share a flavin-binding motif, conserved active-site residuesand are trimeric or dodecameric enzymes. HAL3 proteins preferablycomprise from N-terminus to C-terminus (i) a substrate binding helix,(ii) an insertion His motif, (iii) a PXMNXXMW motif and (iv) a substraterecognition clamp (FIG. 1). These four domains are predicted to beinvolved in substrate binding, the insertion His motif comprises aconserved His residue that is involved in the active site whereas thesequence in the substrate recognition clamp may be somewhat variable insequence and length (Blaesse et al., EMBO J. 19, 6299-6310, 2000). Thestructural features (i) to (iv) are also described in Kupke et al.(2001), which disclosure is incorporated herein by reference. Typically,HAL3 polypeptides are capable of binding of FMN cofactors.

Typically the substrate binding helix is comprised in sequence motif 1with the following consensus sequence (SEQ ID NO: 6):(K/R) PR (V/I) (I/L) LAA (S/T) GSVA (A/S) (I/M/V) KF (G/E/A/S) (N/S/I) L (C/V/A)(H/R/G) (C/S/I) (F/L) (T/S/C) (E/D/Q) WA (E/D) V (R/K) AV (V/A/S).The insertion His motif, the PXMNXXMW motif and the substrate recognitionclamp are part of sequence motif 2 with the following consensus sequence(SEQ ID NO: 7):VLHIELR (R/K/Q) WAD (V/I/A) (L/M) (V/I) IAPLSANTL (G/A) KIAGG (L/M) CDNLLTC(I/V) (I/V) RAWD (Y/F) (T/S/N/D/K) KP (L/F/M/I) F (V/A) APAMNT (L/F) MW(N/S/T) NPFT (E/S/Q/A) (R/K) H (L/F/I) (X₁) (X₂) (L/I/M) (D/N/S) (E/L/Q)(L/M) G (I/V/L) (T/S/A/I) L (I/V) PP (I/V/T) (K/T/S) K (R/T/K) LACGD (Y/H)G (N/T) GAM (A/S) Ewherein X₁ may be any amino acid, preferably X₁ is one of L, V, E, H, D,M, I or Q and wherein X₂ may be any amino acid, preferably X₂ is one of S, L, T, A, or V.

These motifs form part of a larger conserved region in the protein, asan example, the conserved region of Arabidopsis HAL3a is given as SEQ IDNO: 8. HAL3 polypeptides useful in the methods of the present inventionhave in increasing order of preference at least 65%, 70%, 75%, 80%, 85%,90% or 95% sequence identity to the conserved region represented by SEQID NO: 8.

The conserved region in HAL3 proteins, as exemplified in SEQ ID NO: 8and comprising the above described features (i) to (iv), encompasses aFlavoprotein domain which may be identified using specialised databasese.g. SMART (Schultz et al. (1998) Proc. Natl. Acad. Sci. USA 95,5857-5864; Letunic et al. (2002) Nucleic Acids Res 30, 242-244),InterPro (Mulder et al., (2003) Nucl. Acids. Res. 31, 315-318), Prosite(Bucher and Bairoch (1994), A generalized profile syntax forbiomolecular sequences motifs and its function in automatic sequenceinterpretation. (In) ISMB-94; Proceedings 2nd International Conferenceon Intelligent Systems for Molecular Biology. Altman R., Brutlag D.,Karp P., Lathrop R., Searls D., Eds., pp 53-61, AAAIPress, Menlo Park;Hulo et al., Nucl. Acids. Res. 32:D134-D137, (2004)) or Pfam (Bateman etal., Nucleic Acids Research 30(1): 276-280 (2002)). This FMN-bindingdomain (Pfam entry PF02441, InterPro entry IPR003382) is typically foundin flavoprotein enzymes. The terms “domain” and “motif” are defined inthe definitions section above.

The conserved region in SEQ ID NO: 2, as represented by SEQ ID NO: 8,may also in other HAL3 proteins be identified, using methods for thealignment of sequences for comparison as described hereinabove. In someinstances, the default parameters may be adjusted to modify thestringency of the search. For example using BLAST, the statisticalsignificance threshold (called “expect” value) for reporting matchesagainst database sequences may be increased to show less stringentmatches. This way, short nearly exact matches may be identified,including the conserved motifs 1 and 2 (SEQ ID NO: 6 and 7), or matcheswith one or more conservative change at any position.

HAL3 polypeptides (at least in their native form) and their homologuestypically catalyse the decarboxylation of 4′-phosphopantothenoylcysteineto 4′-phosphopantetheine, a step in coenzyme A biosynthesis, whichreaction may be tested in a biochemical assay; alternatively, HAL3activity may be assayed by a complementation test with a dfp mutant E.coli strain (Kupke et al 2001; Yonamine et al 2004). The enzyme is alsoable to decarboxylate pantothenoylcysteine to pantothenoylcysteamine.Furthermore, the protein is involved in conferring salt and osmoticstress tolerance to plants, which feature may be useful in a bioassayfor HAL3.

SEQ ID NO: 2 (encoded by SEQ ID NO: 1) is an example of a HAL3polypeptide comprising from N-terminus to C-terminus features (i) asubstrate binding helix, (ii) an insertion His motif, (iii) a PXMNXXMWmotif and (iv) a substrate recognition clamp; and having in increasingorder of preference at least 65%, 70%, 75%, 80%, 85%, 90% or 95%sequence identity to the conserved region represented by SEQ ID NO: 8.Further examples of HAL3 polypeptides comprising features (i) to (iv)are given in Table A in the examples section below:

Homologues of a HAL3 polypeptide may also be used to perform the methodsof invention. Homologues (or homologous proteins) may readily beidentified using routine techniques well known in the art, such as bysequence alignment. Methods for the alignment of sequences forcomparison are well known in the art, such methods include GAP, BESTFIT,BLAST, FASTA and TFASTA. GAP uses the algorithm of Needleman and Wunsch((1970) J Mol Biol 48: 443-453) to find the alignment of two completesequences that maximizes the number of matches and minimizes the numberof gaps. The BLAST algorithm (Altschul et al. (1990) J Mol Biol 215:403-10) calculates percent sequence identity and performs a statisticalanalysis of the similarity between the two sequences. The software forperforming BLAST analysis is publicly available through the NationalCentre for Biotechnology Information. Homologues may readily beidentified using, for example, the ClustalW multiple sequence alignmentalgorithm (version 1.83), with the default pairwise alignmentparameters, and a scoring method in percentage. Global percentages ofsimilarity and identity may also be determined using one of the methodsavailable in the MatGAT software package (Campanella et al., BMCBioinformatics. 2003 Jul. 10; 4:29. MatGAT: an application thatgenerates similarity/identity matrices using protein or DNA sequences.).Minor manual editing may be performed to optimise alignment betweenconserved motifs, as would be apparent to a person skilled in the art.

The sequence identity values may be determined over the entire conserveddomain (as indicated above) or over the full length nucleic acid oramino acid sequence using the programs mentioned above using the defaultparameters.

Homologues also include orthologues and paralogues. Orthologues andparalogues may easily be found by performing a so-called reciprocalblast search. This may be done by a first BLAST involving submitting aquery sequence (for example, SEQ ID NO: 1 or SEQ ID NO: 2) for a BLASTsearch against any sequence database, such as the publicly availableNCBI database. BLASTN or TBLASTX (using standard default values) may beused when starting from a nucleotide sequence and BLASTP or TBLASTN(using standard default values) may be used when starting from a proteinsequence. The BLAST results may optionally be filtered. The full-lengthsequences of either the filtered results or non-filtered results arethen submitted to a second BLAST search (BLAST back) against sequencesfrom the organism from which the query sequence is derived (where thequery sequence is SEQ ID NO: 1 or SEQ ID NO: 2 the second BLAST wouldtherefore be against Arabidopsis sequences). The results of the firstand second BLAST searches are then compared. A paralogue is identifiedif a high-ranking hit from the first BLAST is from the same species asfrom which the query sequence is derived; an orthologue is identified ifa high-ranking hit is not from the same species as from which the querysequence is derived. Preferred orthologues are orthologues of AtHAL3a(SEQ ID NO: 2), AtHAL3b (SEQ ID NO: 10) or of the long form of AtHAL3b(SEQ ID NO: 40). High-ranking hits are those having a low E-value. Thelower the E-value, the more significant the score (or in other words thelower the chance that the hit was found by chance). Computation of theE-value is well known in the art. In the case of large families,ClustalW may be used, followed by a neighbour joining tree, to helpvisualize clustering of related genes and to identify orthologues andparalogues. In addition to E-values, comparisons are also scored bypercentage identity. Percentage identity refers to the number ofidentical nucleotides (or amino acids) between the two compared nucleicacid (or polypeptide) sequences over a particular length. Preferably,HAL3 polypeptide homologues have in increasing order of preference atleast 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or moresequence identity or similarity (functional identity) to an unmodifiedHAL3 polypeptide as represented by SEQ ID NO: 2. Preferably, HAL3polypeptide homologues are as represented by the sequences referred toin Table A.

The HAL3 polypeptide useful in the methods of the present invention maybe a derivative of SEQ ID NO: 2. “Derivatives” include peptides,oligopeptides, polypeptides which may, compared to the amino acidsequence of the naturally-occurring form of the protein, such as the onepresented in SEQ ID NO: 2, comprise substitutions of amino acids withnon-naturally occurring amino acid residues, or additions ofnon-naturally occurring amino acid residues. Derivatives of the proteinsas represented by the sequences listed in Table A are further exampleswhich may be suitable for use in the methods of the invention

Examples of nucleic acids encoding HAL3 polypeptides include but are notlimited to those represented by the sequences listed in Table A.Variants of nucleic acids encoding HAL3 polypeptides may be suitable foruse in the methods of the invention. Suitable variants include portionsof nucleic acids encoding HAL3 polypeptides and/or nucleic acids capableof hybridising with nucleic acids/genes encoding HAL3 polypeptides.Further variants include splice variants and allelic variants of nucleicacids encoding HAL3 polypeptides.

The term “portion” as defined herein refers to a piece of DNA encoding apolypeptide comprising from N-terminus to C-terminus features (i) asubstrate binding helix, (ii) an insertion His motif, (iii) a PXMNXXMWmotif and (iv) a substrate recognition clamp; and having in increasingorder of preference at least 65%, 70%, 75%, 80%, 85%, 90% or 95%sequence identity to the conserved region represented by SEQ ID NO: 8.

A portion may be prepared, for example, by making one or more deletionsto a nucleic acid encoding a HAL3 polypeptide. The portions may be usedin isolated form or they may be fused to other coding (or non coding)sequences in order to, for example, produce a protein that combinesseveral activities. When fused to other coding sequences, the resultingpolypeptide produced upon translation may be bigger than that predictedfor the HAL3 portion. Preferably, the portion codes for a polypeptidewith substantially the same biological activity as the HAL3 polypeptideof SEQ ID NO: 2. The portion is typically at least 50, 100, 150 or 200nucleotides in length, preferably at least 250, 300, 350 or 400nucleotides in length, more preferably at least 450, 500, 550, 600 or650 nucleotides in length.

Preferably, the portion is a portion of a nucleic acid as represented bythe sequences listed in Table A. Most preferably the portion is aportion of a nucleic acid as represented by SEQ ID NO: 1.

The terms “fragment”, “fragment of a sequence” or “part of a sequence”“portion” or “portion thereof” mean a truncated sequence of the originalsequence referred to. The truncated sequence (nucleic acid or proteinsequence) can vary widely in length; the minimum size being a sequenceof sufficient size to provide a sequence with at least a comparablefunction and/or activity of the original sequence referred to orhybidizing with the nucleic acid molecule of the invention or used inthe process of the invention under stringent conditions, while themaximum size is not critical. In some applications, the maximum sizeusually is not substantially greater than that required to provide thedesired activity and/or function(s) of the original sequence. Acomparable function means at least 40%, 45% or 50%, preferably at least60%, 70%, 80% or 90% or more of the original sequence.

Another variant of a nucleic acid encoding a HAL3 polypeptide, useful inthe methods of the present invention, is a nucleic acid capable ofhybridising under reduced stringency conditions, preferably understringent conditions, with a probe derived from the nucleic acid asdefined hereinbefore, which hybridising sequence encodes a polypeptidecomprising from N-terminus to C-terminus features (i) a substratebinding helix, (ii) an insertion His motif, (iii) a PXMNXXMW motif and(iv) a substrate recognition clamp; and has in increasing order ofpreference at least 65%, 70%, 75%, 80%, 85%, 90% or 95% sequenceidentity to the conserved region represented by SEQ ID NO: 8.

Preferably, the hybridising sequence is one that is capable ofhybridising to a nucleic acid as represented by (or to probes derivedfrom) the sequences listed in Table A, or to a portion of any of thesesequences (the target sequence). Most preferably the hybridisingsequence is capable of hybridising to SEQ ID NO: 1 (or to probes derivedtherefrom). Probes are generally less than 700 bp or 600 bp in length,preferably less than 500, 400 bp, 300 bp 200 bp or 100 bp in length.Commonly, probe lengths for DNA-DNA hybridisations such as Southernblotting, vary between 100 and 500 bp, whereas the hybridising region inprobes for DNA-DNA hybridisations such as in PCR amplification generallyare shorter than 50 but longer than 10 nucleotides, preferably they are15, 20, 25, 30, 35, 40, 45 or 50 bp in length.

The HAL3 polypeptide may be encoded by a splice variant. The term“splice variant” as used herein encompasses variants of a nucleic acidsequence in which selected introns and/or exons have been excised,replaced, displaced or added, or in which introns have been shortened orlengthened. Such variants will be ones in which the substantialbiological activity of the protein is retained, which may be achieved byselectively retaining functional segments of the protein. Such splicevariants may be found in nature or may be manmade. Methods for makingsuch splice variants are well known in the art.

Preferred splice variants are splice variants of the nucleic acidencoding a HAL3 polypeptide comprising from N-terminus to C-terminus (i)a substrate binding helix, (ii) an insertion His motif, (iii) a PXMNXXMWmotif and (iv) a substrate recognition clamp; and having in increasingorder of preference at least 65%, 70%, 75%, 80%, 85%, 90% or 95%sequence identity to the conserved region represented by SEQ ID NO: 8.

Further preferred splice variants of nucleic acids encoding HAL3polypeptides comprising features as defined hereinabove are splicevariants of a nucleic acid as represented by any one of the sequenceslisted in Table A. Most preferred is a splice variant of a nucleic acidsequence as represented by SEQ ID NO: 1.

The HAL3 polypeptide may also be encoded by an allelic variant of anucleic acid encoding a polypeptide comprising from N-terminus toC-terminus (i) a substrate binding helix, (ii) an insertion His motif,(iii) a PXMNXXMW motif and (iv) a substrate recognition clamp; andhaving in increasing order of preference at least 65%, 70%, 75%, 80%,85%, 90% or 95% sequence identity to the conserved region represented bySEQ ID NO: 8.

Preferred allelic variants of nucleic acids encoding HAL3 polypeptidescomprising features as defined hereinabove are allelic variants of anucleic acid as represented by any one of the sequences listed in TableA. Most preferred is an allelic variant of a nucleic acid sequence asrepresented by SEQ ID NO: 1.

Directed evolution (or gene shuffling) may also be used to generatevariants of nucleic acids encoding HAL3 polypeptides. Site-directedmutagenesis may be used to generate variants of nucleic acids encodingHAL3 polypeptides. Several methods are available to achievesite-directed mutagenesis, the most common being PCR based methods(Current Protocols in Molecular Biology. Wiley Eds.).

Nucleic acids encoding HAL3 polypeptides may be derived from any naturalor artificial source. The nucleic acid may be modified from its nativeform in composition and/or genomic environment through deliberate humanmanipulation. Preferably the HAL3 nucleic acid is from a plant, furtherpreferably from a dicotyledonous plant, more preferably from the familyBrassicaceae, most preferably the nucleic acid is from Arabidopsisthaliana.

The increased expression of a nucleic acid encoding a HAL3 polypeptide,preferentially in shoots of a plant, may be performed by introducing agenetic modification (preferably in the locus of a HAL3 gene). The locusof a gene as defined herein is taken to mean a genomic region, whichincludes the gene of interest and 10 KB up- or downstream of the codingregion.

The genetic modification may be introduced, for example, by any one (ormore) of the following methods: T-DNA activation, TILLING and homologousrecombination (as described in the definitions section) or byintroducing and increasing expression a nucleic acid encoding a HAL3polypeptide preferentially in expanding tissues of a plant. Followingintroduction of the genetic modification, there follows an optional stepof selecting for increased expression of a nucleic acid encoding a HAL3factor polypeptide preferentially in expanding tissues, which increasedexpression gives plants having increased yield.

T-DNA activation tagging results in transgenic plants that show dominantphenotypes due to modified expression of genes close to the introducedpromoter. The promoter to be introduced may be any promoter capable ofincreasing expression of the nucleic acid encoding a HAL3 polypeptidepreferentially in expanding tissues of a plant.

A genetic modification may also be introduced in the locus of a geneencoding a HAL3 polypeptide using the technique of TILLING (TargetedInduced Local Lesions In Genomes). Plants carrying such mutant variantshave increased expression of a nucleic acid encoding a HAL3 polypeptidepreferentially in expanding plant tissues.

T-DNA activation and TILLING are examples of technologies that enablethe generation of genetic modifications comprising preferentiallyincreasing expression of a nucleic acid encoding a HAL3 polypeptide inexpanding tissues of plants.

The effects of the invention may also be reproduced using homologousrecombination. The nucleic acid to be targeted is preferably the regioncontrolling the natural expression of a nucleic acid encoding a HAL3polypeptide in a plant. A weak promoter specific for expression inshoots is introduced into this region, replacing it partly orsubstantially all of it.

A preferred method for introducing a genetic modification (which in thiscase need not be in the locus of a HAL3 gene) is to introduce andexpress preferentially in the shoot of a plant a nucleic acid encoding aHAL3 polypeptide, as defined hereinabove. The nucleic acid to beintroduced into a plant may be a full-length nucleic acid or may be aportion or a hybridising sequence or another nucleic acid variant ashereinbefore defined.

The methods of the invention rely on preferentially increased expressionof a nucleic acid encoding a HAL3 polypeptide in shoot tissue of aplant, preferably in the cell expansion zone of vegetative shoots.

The invention also provides genetic constructs and vectors to facilitateintroduction and/or preferential expression of the nucleic acidsequences useful in the methods according to the invention, in shoots,preferably in expanding tissues of a plant shoots.

Therefore, there is provided a gene construct comprising:

-   -   (i) a nucleic acid encoding a HAL3 polypeptide as defined        hereinabove;    -   (ii) one or more control sequences, of which at least one is a        shoot-specific promoter, operably linked to the nucleic acid of        (i).

Constructs useful in the methods according to the present invention maybe constructed using recombinant DNA technology well known to personsskilled in the art. The gene constructs may be inserted into vectors,which may be commercially available, suitable for transforming intoplants and suitable for expression of the gene of interest in thetransformed cells. The invention therefore provides use of a geneconstruct as defined hereinabove in the methods of the invention.

Plants are transformed with a vector comprising the sequence of interest(i.e., a nucleic acid encoding a HAL3 polypeptide). The skilled artisanis well aware of the genetic elements that must be present on the vectorin order to successfully transform, select and propagate host cellscontaining the sequence of interest. The sequence of interest isoperably linked to one or more control sequences (at least to apromoter).

Suitable promoters, which are functional in plants, are generally known.They may take the form of constitutive or inducible promoters. Suitablepromoters can enable the development- and/or tissue-specific expressionin multi-celled eukaryotes; thus, leaf-, root-, flower-, seed-,stomata-, tuber- or fruit-specific promoters may advantageously be usedin plants.

Different plant promoters usable in plants are promoters such as, forexample, the USP, the LegB4-, the DC3 promoter or the ubiquitin promoterfrom parsley.

For expression in plants, the nucleic acid molecule must, as describedabove, be linked operably to or comprise a suitable promoter whichexpresses the gene at the right point in time and in a cell- ortissue-specific manner. Usable promoters are constitutive promoters(Benfey et al., EMBO J. 8 (1989) 2195-2202), such as those whichoriginate from plant viruses, such as 35S CAMV (Franck et al., Cell 21(1980) 285-294), 19S CaMV (see also U.S. Pat. No. 5,352,605 and WO84/02913), 34S FMV (Sanger et al., Plant. Mol. Biol., 14, 1990:433-443), the parsley ubiquitin promoter, or plant promoters such as theRubisco small subunit promoter described in U.S. Pat. No. 4,962,028 orthe plant promoters PRP1 [ Ward et al., Plant. Mol. Biol. 22 (1993)],SSU, PGEL1, OCS [Leisner (1988) Proc Natl Acad Sci USA 85(5):2553-2557], lib4, usp, mas [Comai (1990) Plant Mol Biol 15 (3):373-381],STLS1, ScBV (Schenk (1999) Plant Mol Biol 39(6):1221-1230), B33, SAD1 orSAD2 (flax promoters, Jain et al., Crop Science, 39 (6), 1999:1696-1701) or nos [Shaw et al. (1984) Nucleic Acids Res.12(20):7831-7846]. Further examples of constitutive plant promoters arethe sugarbeet V-ATPase promoters (WO 01/14572). Examples of syntheticconstitutive promoters are the Super promoter (WO 95/14098) andpromoters derived from G-boxes (WO 94/12015). If appropriate, chemicalinducible promoters may furthermore also be used, compare EP-A 388186,EP-A 335528, WO 97/06268. Stable, constitutive expression of theproteins according to the invention a plant can be advantageous.However, inducible expression of the polypeptide of the invention isadvantageous, if a late expression before the harvest is of advantage,as metabolic manipulation may lead to plant growth retardation.

The expression of plant genes can also be facilitated via a chemicalinducible promoter (for a review, see Gatz 1997, Annu. Rev. PlantPhysiol. Plant Mol. Biol., 48:89-108). Chemically inducible promotersare particularly suitable when it is desired to express the gene in atime-specific manner. Examples of such promoters are a salicylic acidinducible promoter (WO 95/19443), and abscisic acid-inducible promoter(EP 335 528), a tetracyclin-inducible promoter (Gatz et al. (1992) PlantJ. 2, 397-404), a cyclohexanol- or ethanol-inducible promoter (WO93/21334) or others as described herein.

Other suitable promoters are those which react to biotic or abioticstress conditions, for example the pathogen-induced PRP1 gene promoter(Ward et al., Plant. Mol. Biol. 22 (1993) 361-366), the tomatoheat-inducible hsp80 promoter (U.S. Pat. No. 5,187,267), the potatochill-inducible alpha-amylase promoter (WO 96/12814) or thewound-inducible pinII promoter (EP-A-0 375 091) or others as describedherein.

Preferred promoters are in particular those which bring gene expressionin tissues and organs, in seed cells, such as endosperm cells and cellsof the developing embryo. Suitable promoters are the oilseed rape napingene promoter (U.S. Pat. No. 5,608,152), the Vicia faba USP promoter(Baeumlein et al., Mol Gen Genet, 1991, 225 (3): 459-67), theArabidopsis oleosin promoter (WO 98/45461), the Phaseolus vulgarisphaseolin promoter (U.S. Pat. No. 5,504,200), the Brassica Bce4 promoter(WO 91/13980), the bean arc5 promoter, the carrot DcG3 promoter, or theLegumin B4 promoter (LeB4; Baeumlein et al., 1992, Plant Journal, 2 (2):233-9), and promoters which bring about the seed-specific expression inmonocotyledonous plants such as maize, barley, wheat, rye, rice and thelike. Advantageous seed-specific promoters are the sucrose bindingprotein promoter (WO 00/26388), the phaseolin promoter and the napinpromoter. Suitable promoters which must be considered are the barleyIpt2 or Ipt1 gene promoter (WO 95/15389 and WO 95/23230), and thepromoters described in WO 99/16890 (promoters from the barley hordeingene, the rice glutelin gene, the rice oryzin gene, the rice prolamingene, the wheat gliadin gene, the wheat glutelin gene, the maize zeingene, the oat glutelin gene, the sorghum kasirin gene and the ryesecalin gene). Further suitable promoters are Amy32b, Amy 6-6 andAleurain [U.S. Pat. No. 5,677,474], Bce4 (oilseed rape) [U.S. Pat. No.5,530,149], glycinin (soya) [EP 571 741], phosphoenolpyruvatecarboxylase (soya) [JP 06/62870], ADR12-2 (soya) [WO 98/08962],isocitrate lyase (oilseed rape) [U.S. Pat. No. 5,689,040] or α-amylase(barley) [EP 781 849]. Other promoters which are available for theexpression of genes in plants are leaf-specific promoters such as thosedescribed in DE-A 19644478 or light-regulated promoters such as, forexample, the pea petE promoter.

Further suitable plant promoters are the cytosolic FBPase promoter orthe potato ST-LSI promoter (Stockhaus et al., EMBO J. 8, 1989, 2445),the Glycine max phosphoribosylpyrophosphate amidotransferase promoter(GenBank Accession No. U87999) or the node-specific promoter describedin EP-A-0 249 676.

In a preferred embodiment, the nucleic acid sequence encoding a HAL3polypeptide is operably linked to a shoot a shoot specific promoter. Ashoot-specific promoter refers to any promoter able to drive expressionof the gene of interest in vegetative plant shoot tissues. Preferably,the shoot specific promoter is able to preferentially drive expressionof the gene of interest in expanding tissues of vegetative shoots, thatis in the cell expansion zone of vegetative shoots. Reference herein to“preferentially” driving expression in the shoot is taken to meandriving expression of any sequence operably linked thereto in the cellexpansion zone of vegetative shoots substantially to the exclusion ofdriving expression elsewhere in the plant, apart from any residualexpression due to leaky promoter expression. The shoot-specific promotermay be either a natural or a synthetic promoter.

Preferably, the shoot-specific promoter is a promoter isolated from abeta-expansin gene, such as a rice beta expansin EXBP9 promoter (WO2004/070039), as represented by SEQ ID NO: 5 or a promoter of similarstrength and/or a promoter with a similar expression pattern as the ricebeta expansin promoter (i.e. a functionally equivalent promoter).

It should be clear that the applicability of the present invention isnot restricted to the nucleic acid encoding a HAL3 polypeptiderepresented by SEQ ID NO: 1, nor is the applicability of the inventionrestricted to expression of a nucleic acid encoding a HAL3 polypeptidewhen driven by a beta expansin promoter.

Optionally, one or more terminator sequences (also a control sequence)may be used in the construct introduced into a plant. Additionalregulatory elements may include transcriptional as well as translationalenhancers. Those skilled in the art will be aware of terminator andenhancer sequences that may be suitable for use in performing theinvention. Such sequences would be known or may readily be obtained by aperson skilled in the art.

The genetic constructs of the invention may further include an origin ofreplication sequence that is required for maintenance and/or replicationin a specific cell type. One example is when a genetic construct isrequired to be maintained in a bacterial cell as an episomal geneticelement (e.g. plasmid or cosmid molecule). Preferred origins ofreplication include, but are not limited to, the f1-ori and colE1.

Other control sequences (besides promoter, enhancer, silencer, intronsequences, 3′UTR and/or 5′UTR regions) may be protein and/or RNAstabilizing elements.

For the detection and/or selection of the successful transfer of thenucleic acid sequences as depicted in the sequence protocol and used inthe process of the invention, it is advantageous to use marker genes(=reporter genes). These marker genes enable the identification of asuccessful transfer of the nucleic acid molecules via a series ofdifferent principles, for example via visual identification with the aidof fluorescence, luminescence or in the wavelength range of light whichis discernible for the human eye, by a resistance to herbicides orantibiotics, via what are known as nutritive markers (auxotrophismmarkers) or antinutritive markers, via enzyme assays or viaphytohormones. Examples of such markers which may be mentioned are GFP(=green fluorescent protein); the luciferin/luceferase system, theβ-galactosidase with its colored substrates, for example X-Gal, theherbicide resistances to, for example, imidazolinone, glyphosate,phosphinothricin or sulfonylurea, the antibiotic resistances to, forexample, bleomycin, hygromycin, streptomycin, kanamycin, tetracyclin,chloramphenicol, ampicillin, gentamycin, geneticin (G418), spectinomycinor blasticidin, to mention only a few, nutritive markers such as theutilization of mannose or xylose, or antinutritive markers such as theresistance to 2-deoxyglucose. This list is a small number of possiblemarkers. The skilled worker is very familiar with such markers.Different markers are preferred, depending on the organism and theselection method.

The present invention also encompasses plants obtainable by the methodsaccording to the present invention. The present invention thereforeprovides plants, plant parts or plant cells thereof obtainable by themethod according to the present invention, which plants or parts orcells thereof comprise a nucleic acid transgene encoding a HAL3polypeptide under the control of a shoot-specific promoter.

The invention also provides a method for the production of transgenicplants having increased yield relative to control plants, comprisingintroduction and preferential expression of a nucleic acid encoding aHAL3 polypeptide in the shoot of a plant.

Host plants for the nucleic acids or the vector used in the methodaccording to the invention, the expression cassette or construct orvector are, in principle, advantageously all plants, which are capableof synthesizing the polypeptides used in the inventive method.

More specifically, the present invention provides a method for theproduction of transgenic plants having increased yield which methodcomprises:

-   -   (i) introducing and preferentially expressing a nucleic acid        encoding a HAL3 polypeptide in the shoot of a plant; and    -   (ii) cultivating the plant cell under conditions promoting plant        growth and development.

The nucleic acid may be introduced directly into a plant cell or intothe plant itself (including introduction into a tissue, organ or anyother part of a plant). According to a preferred feature of the presentinvention, the nucleic acid is preferably introduced into a plant bytransformation.

The transfer of foreign genes into the genome of a plant is calledtransformation. In doing this the methods described for thetransformation and regeneration of plants from plant tissues or plantcells are utilized for transient or stable transformation. To selecttransformed plants, the plant material obtained in the transformationis, as a rule, subjected to selective conditions so that transformedplants can be distinguished from untransformed plants. For example, theseeds obtained in the above-described manner can be planted and, afteran initial growing period, subjected to a suitable selection byspraying. A further possibility consists in growing the seeds, ifappropriate after sterilization, on agar plates using a suitableselection agent so that only the transformed seeds can grow into plants.Further advantageous transformation methods, in particular for plants,are known to the skilled worker and are described herein below.

Generally after transformation, plant cells or cell groupings areselected for the presence of one or more markers which are encoded byplant-expressible genes co-transferred with the gene of interest,following which the transformed material is regenerated into a wholeplant.

As mentioned Agrobacteria transformed with an expression vectoraccording to the invention may also be used in the manner known per sefor the transformation of plants such as experimental plants likeArabidopsis or crop plants, such as, for example, cereals, maize, oats,rye, barley, wheat, soya, rice, cotton, sugarbeet, canola, sunflower,flax, hemp, potato, tobacco, tomato, carrot, bell peppers, oilseed rape,tapioca, cassava, arrow root, tagetes, alfalfa, lettuce and the varioustree, nut, and grapevine species, in particular oil-containing cropplants such as soya, peanut, castor-oil plant, sunflower, maize, cotton,flax, oilseed rape, coconut, oil palm, safflower (Carthamus tinctorius)or cocoa beans, for example by bathing scarified leaves or leaf segmentsin an agrobacterial solution and subsequently growing them in suitablemedia.

In addition to the transformation of somatic cells, which then has to beregenerated into intact plants, it is also possible to transform thecells of plant meristems and in particular those cells which developinto gametes. In this case, the transformed gametes follow the naturalplant development, giving rise to transgenic plants. Thus, for example,seeds of Arabidopsis are treated with agrobacteria and seeds areobtained from the developing plants of which a certain proportion istransformed and thus transgenic [Feldman, K A and Marks M D (1987). MolGen Genet. 208:274-289; Feldmann K (1992). In: C Koncz, N-H Chua and JShell, eds, Methods in Arabidopsis Research. Word Scientific, Singapore,pp. 274-289]. Alternative methods are based on the repeated removal ofthe influorescences and incubation of the excision site in the center ofthe rosette with transformed agrobacteria, whereby transformed seeds canlikewise be obtained at a later point in time (Chang (1994). Plant J. 5:551-558; Katavic (1994). Mol Gen Genet, 245: 363-370). However, anespecially effective method is the vacuum infiltration method with itsmodifications such as the “floral dip” method. In the case of vacuuminfiltration of Arabidopsis, intact plants under reduced pressure aretreated with an agrobacterial suspension [Bechthold, N (1993). C R AcadSci Paris Life Sci, 316: 1194-1199], while in the case of the“floraldip” method the developing floral tissue is incubated briefly with asurfactant-treated agrobacterial suspension [Clough, S J and Bent, A F(1998). The Plant J. 16, 735-743]. A certain proportion of transgenicseeds are harvested in both cases, and these seeds can be distinguishedfrom nontransgenic seeds by growing under the above-described selectiveconditions. In addition the stable transformation of plastids is ofadvantages because plastids are inherited maternally is most cropsreducing or eliminating the risk of transgene flow through pollen. Thetransformation of the chloroplast genome is generally achieved by aprocess, which has been schematically displayed in Klaus et al., 2004 [Nature Biotechnology 22 (2), 225-229]. Briefly the sequences to betransformed are cloned together with a selectable marker gene betweenflanking sequences homologous to the chloroplast genome. Thesehomologous flanking sequences direct site specific integration into theplastome. Plastidal transformation has been described for many differentplant species and an overview can be taken from Bock (2001) Transgenicplastids in basic research and plant biotechnology. J Mol Biol. 2001Sep. 21; 312 (3):425-38 or Maliga, P (2003) Progress towardscommercialization of plastid transformation technology. TrendsBiotechnol. 21, 20-28. Further biotechnological progress has recentlybeen reported in form of marker free plastid transformants, which can beproduced by a transient cointegrated maker gene (Klaus et al., 2004,Nature Biotechnology 22(2), 225-229).

The genetically modified plant cells can be regenerated via all methodswith which the skilled worker is familiar. Suitable methods can be foundin the abovementioned publications by S. D. Kung and R. Wu, Potrykus orHöfgen and Willmitzer.

Following DNA transfer and regeneration, putatively transformed plantsmay be evaluated, for instance using Southern analysis, for the presenceof the gene of interest, copy number and/or genomic organisation.Alternatively or additionally, expression levels of the newly introducedDNA may be monitored using Northern and/or Western analysis, orquantitative PCR, all techniques being well known to persons havingordinary skill in the art.

The generated transformed plants may be propagated by a variety ofmeans, such as by clonal propagation or classical breeding techniques.For example, a first generation (or T1) transformed plant may be selfedto give homozygous second generation (or T2) transformants, and the T2plants further propagated through classical breeding techniques.

The generated transformed organisms may take a variety of forms. Forexample, they may be chimeras of transformed cells and non-transformedcells; clonal transformants (e.g., all cells transformed to contain theexpression cassette); grafts of transformed and untransformed tissues(e.g., in plants, a transformed rootstock grafted to an untransformedscion).

The present invention clearly extends to any plant cell or plantproduced by any of the methods described herein, and to all plant partsand propagules thereof. The present invention extends further toencompass the progeny of a primary transformed or transfected cell,tissue, organ or whole plant that has been produced by any of theaforementioned methods, the only requirement being that progeny exhibitthe same genotypic and/or phenotypic characteristic(s) as those producedby the parent in the methods according to the invention.

The invention also includes host cells containing an isolated nucleicacid encoding a HAL3 polypeptide. Preferred host cells according to theinvention are plant cells.

The invention also extends to harvestable parts of a plant such as, butnot limited to seeds, leaves, fruits, flowers, stems, rhizomes, tubersand bulbs. The invention furthermore relates to products derived,preferably directly derived, from a harvestable part of such a plant,such as dry pellets or powders, oil, fat and fatty acids, starch orproteins.

The present invention also encompasses use of nucleic acids encodingHAL3 polypeptides and use of HAL3 polypeptides in increasing plant yieldas defined hereinabove in the methods of the invention.

Nucleic acids encoding HAL3 polypeptides, or HAL3 polypeptides, may finduse in breeding programmes in which a DNA marker is identified which maybe genetically linked to a HAL3 gene. The nucleic acids/genes, or theHAL3 polypeptides may be used to define a molecular marker. This DNA orprotein marker may then be used in breeding programmes to select plantshaving increased yield as defined hereinabove in the methods of theinvention.

Allelic variants of a HAL3 nucleic acid/gene may also find use inmarker-assisted breeding programmes. Such breeding programmes sometimesrequire introduction of allelic variation by mutagenic treatment of theplants, using for example EMS mutagenesis; alternatively, the programmemay start with a collection of allelic variants of so called “natural”origin caused unintentionally. Identification of allelic variants thentakes place, for example, by PCR. This is followed by a step forselection of superior allelic variants of the sequence in question andwhich give increased yield. Selection is typically carried out bymonitoring growth performance of plants containing different allelicvariants of the sequence in question. Growth performance may bemonitored in a greenhouse or in the field. Further optional stepsinclude crossing plants in which the superior allelic variant wasidentified with another plant. This could be used, for example, to makea combination of interesting phenotypic features.

A nucleic acid encoding a HAL3 polypeptide may also be used as probesfor genetically and physically mapping the genes that they are a partof, and as markers for traits linked to those genes. Such informationmay be useful in plant breeding in order to develop lines with desiredphenotypes. Such use of HAL3 nucleic acids requires only a nucleic acidsequence of at least 15 nucleotides in length. The HAL3 nucleic acidsmay be used as restriction fragment length polymorphism (RFLP) markers.Southern blots (Sambrook J, Fritsch E F and Maniatis T (1989) MolecularCloning, A Laboratory Manual) of restriction-digested plant genomic DNAmay be probed with the HAL3 nucleic acids. The resulting bandingpatterns may then be subjected to genetic analyses using computerprograms such as MapMaker (Lander et al. (1987) Genomics 1: 174-181) inorder to construct a genetic map. In addition, the nucleic acids may beused to probe Southern blots containing restriction endonuclease-treatedgenomic DNAs of a set of individuals representing parent and progeny ofa defined genetic cross. Segregation of the DNA polymorphisms is notedand used to calculate the position of the HAL3 nucleic acid in thegenetic map previously obtained using this population (Botstein et al.(1980) Am. J. Hum. Genet. 32:314-331).

The production and use of plant gene-derived probes for use in geneticmapping is described in Bernatzky and Tanksley (1986) Plant Mol. Biol.Reporter 4: 37-41. Numerous publications describe genetic mapping ofspecific cDNA clones using the methodology outlined above or variationsthereof. For example, F2 intercross populations, backcross populations,randomly mated populations, near isogenic lines, and other sets ofindividuals may be used for mapping. Such methodologies are well knownto those skilled in the art.

The nucleic acid probes may also be used for physical mapping (i.e.,placement of sequences on physical maps; see Hoheisel et al. In:Non-mammalian Genomic Analysis: A Practical Guide, Academic press 1996,pp. 319-346, and references cited therein).

In another embodiment, the nucleic acid probes may be used in directfluorescence in situ hybridisation (FISH) mapping (Trask (1991) TrendsGenet. 7:149-154). Although current methods of FISH mapping favour useof large clones (several kb to several hundred kb; see Laan et al.(1995) Genome Res. 5:13-20), improvements in sensitivity may allowperformance of FISH mapping using shorter probes.

A variety of nucleic acid amplification-based methods for genetic andphysical mapping may be carried out using the nucleic acids. Examplesinclude allele-specific amplification (Kazazian (1989) J. Lab. Clin. Med11:95-96), polymorphism of PCR-amplified fragments (CAPS; Sheffield etal. (1993) Genomics 16:325-332), allele-specific ligation (Landegren etal. (1988) Science 241:1077-1080), nucleotide extension reactions(Sokolov (1990) Nucleic Acid Res. 18:3671), Radiation Hybrid Mapping(Walter et al. (1997) Nat. Genet. 7:22-28) and Happy Mapping (Dear andCook (1989) Nucleic Acid Res. 17:6795-6807). For these methods, thesequence of a nucleic acid is used to design and produce primer pairsfor use in the amplification reaction or in primer extension reactions.The design of such primers is well known to those skilled in the art. Inmethods employing PCR-based genetic mapping, it may be necessary toidentify DNA sequence differences between the parents of the mappingcross in the region corresponding to the instant nucleic acid sequence.This, however, is generally not necessary for mapping methods.

The methods according to the present invention result in plants havingincreased yield, as described hereinbefore. This increased yield mayalso be combined with other economically advantageous traits, such asfurther yield-enhancing traits, tolerance to other abiotic and bioticstresses, traits modifying various architectural features and/orbiochemical and/or physiological features.

Detailed Description for MADS15 up

Surprisingly, it has now been found that modulating expression in aplant of a nucleic acid encoding a MADS15 polypeptide gives plantshaving enhanced yield-related traits relative to control plants. Theparticular class of MADS15 polypeptides suitable for enhancingyield-related traits in plants is described in detail below.

The present invention provides a method for enhancing yield-relatedtraits in plants relative to control plants, comprising modulatingexpression in a plant of a nucleic acid encoding a MADS15 polypeptide.

Any reference hereinafter to a “protein useful in the methods of theinvention” is taken to mean a MADS15 polypeptide as defined herein. Anyreference hereinafter to a “nucleic acid useful in the methods of theinvention” is taken to mean a nucleic acid capable of encoding such aMADS15 polypeptide.

A preferred method for modulating (preferably, increasing) expression ofa nucleic acid encoding a protein useful in the methods of the inventionis by introducing and expressing in a plant a nucleic acid encoding aprotein useful in the methods of the invention as defined below.

The nucleic acid to be introduced into a plant (and therefore useful inperforming the methods of the invention) is any nucleic acid encodingthe type of protein which will now be described, hereafter also named“MADS15 nucleic acid” or “MADS15 gene”.

The term “MADS15 polypeptide” as defined herein refers to a protein thatfalls in the group of FUL-like MADS box proteins as delineated by Adamet al. (J. Mol. Evol. 62, 15-31). FUL-like MADS box proteins are part ofthe SQUAMOSA subfamily and have the typical MIKC^(c) architecture: aMADS domain (M) at the N-terminus, followed by an intervening domain (I)involved in dimerisation, a highly conserved keratin domain (K) and avariable domain (C) at the C-terminus, which is responsible for bindingof interacting proteins or transcriptional activation (De Bodt et al.Trends in Plant Science 8, 475-483).

Plant MADS15 polypeptides may also be identified by the presence ofcertain conserved motifs. The presence of these conserved motifs may beidentified using methods for the alignment of sequences for comparisonas described hereinabove. In some instances, the default parameters maybe adjusted to modify the stringency of the search. For example usingBLAST, the statistical significance threshold (called “expect” value)for reporting matches against database sequences may be increased toshow less stringent matches. This way, short nearly exact matches may beidentified. Upon identification of a MADS15 polypeptide by the presenceof these motifs, a person skilled in the art may easily derive thecorresponding nucleic acid encoding the polypeptide comprising therelevant motifs, and use a sufficient length of contiguous nucleotidesof the same to perform any one or more of the gene silencing methodsdescribed above (for the reduction or substantial elimination of anendogenous MADS15 gene expression).

Typically, the presence of at least one of the motifs 1 to 4 should besufficient to identify any query sequence as a MADS15, however, thepresence of at least motifs 1 and 2 is preferred. The consensus sequenceprovided is based on the monocot sequences given in the sequencelisting. A person skilled in the art would be well aware that theconsensus sequence may vary somewhat if further or different sequences(for example from dicotyledonous plants) were used for comparison.

Motif 1, located in the MADS domain (SEQ ID NO: 48):L (L/V) KKA (H/N) EIS (VI) L (C/Y) DAE (V/I/L) (A/G) (L/A/V) (I/V) (I /V) FS(T/P/A/N) KGKLYE (Y/F) (A/S) (T/S) (D/N/E) S (K/C/R/S) M (D/E) (N/I/R/K) IL(E/D) R. Preferably, this conserved sequence motif 1 has the sequence:LLKKAHEISVLCDAEVA (L/A/V) I (I/V) FS (T/P) KGKLYEYATDS (C/R) M (D/E) (R/K)ILER, most preferably the conserved sequence motif 1 has the sequence:LLKKAHEISVLCDAEVAAIVFSPKGKLYEYATDSRMDKILERMotif 2, located in the K domain (SEQ ID NO: 49):KLK (A/S) (K/R) (V/I ) E (A/T/S) (L/I) (Q/N) (K/R/N) (S/C/R) (Q/H) (R/K)HLMGE Preferably, this conserved sequence motif 2 has the sequence:KLKAK (V/I) E (A/T) (L/I) QK (S/C) (Q/H) (R/K) HLMGEMore preferably, this conserved sequence motif 2 has the sequence:KLKAKIETIQKCHKHLMGE

Optionally, the MADS15 polypeptide or its homologue may comprise in theC domain motif 3 and/or motif 4:

motif 3 (SEQ ID NO: 50): Q (P/Q/V/A) QTS (S/F) (S/F) (S/F) (S/F)(S/C/F) (F/M) motif 4 (SEQ ID NO: 51): (G/A/V/L) (L/P) XWMX (S/H)wherein the first X residue in motif 4 may be any amino acid, butpreferably L, P or H; and wherein the second X residue may be any aminoacid, but preferably V or L.

Alternatively, the C-domain (starting behind the keratin domain, i.e. atR175 in SEQ ID NO: 44) may be characterised by the increased occurrenceof glutamine (normally 3.93%, here at least 8.77% with a maximum of26.73%), in addition, the content in alanine, proline, and/or serine mayalso be increased (above 7.8%, 4.85% and 6.89% respectively).

Alternatively formulated, a preferred MADS15 protein useful in themethods of the present invention comprises at least an N-terminal MADSdomain corresponding to the SMART domain SM00432 (Pfam PF00319) followedby a Keratin domain corresponding to the K-box region defined in Pfam asPF01486, more preferably, the MADS15 protein has in its MADS domain theconserved signature of SEQ ID NO: 48 and in its K-domain the conservedsignature of SEQ ID NO: 49, most preferably, the MADS15 protein has thesequence given in SEQ ID NO: 44.

Examples of proteins useful in the methods of the invention and nucleicacids encoding the same are given below in table D of Example 8.

Also useful in the methods of the invention are homologues of any one ofthe amino acid sequences given in table D.

Also useful in the methods of the invention are derivatives of any oneof the polypeptides given in table D or orthologues or paralogues of anyof the aforementioned SEQ ID NOs. A preferred derivative is a derivativeof SEQ ID NO: 44. Derivatives of the polypeptides given in table D arefurther examples which may be suitable for use in the methods of theinvention. Derivatives useful in the methods of the present inventionpreferably have similar biological and functional activity as theunmodified protein from which they are derived.

The invention is illustrated by transforming plants with the Oryzasativa nucleic acid sequence represented by SEQ ID NO: 43, encoding thepolypeptide sequence of SEQ ID NO: 44, however performance of theinvention is not restricted to these sequences. The methods of theinvention may advantageously be performed using any nucleic acidencoding a protein useful in the methods of the invention as definedherein, including orthologues and paralogues, such as any of the nucleicacid sequences given in table D. The amino acid sequences given in tableD may be considered to be orthologues and paralogues of the MADS15polypeptide represented by SEQ ID NO: 44.

Orthologues and paralogues may easily be found by performing a so-calledreciprocal blast search. Typically, this involves a first BLASTinvolving BLASTing a query sequence (for example using any of thesequences listed in table D) against any sequence database, such as thepublicly available NCBI database. BLASTN or TBLASTX (using standarddefault values) are generally used when starting from a nucleotidesequence, and BLASTP or TBLASTN (using standard default values) whenstarting from a protein sequence. The BLAST results may optionally befiltered. The full-length sequences of either the filtered results ornon-filtered results are then BLASTed back (second BLAST) againstsequences from the organism from which the query sequence is derived(where the query sequence is SEQ ID NO: 43 or SEQ ID NO: 44, the secondBLAST would therefore be against Oryza sativa sequences). The results ofthe first and second BLASTs are then compared. A paralogue is identifiedif a high-ranking hit from the first blast is from the same species asfrom which the query sequence is derived, a BLAST back then ideallyresults in the query sequence as highest hit; an orthologue isidentified if a high-ranking hit in the first BLAST is not from the samespecies as from which the query sequence is derived, and preferablyresults upon BLAST back in the query sequence being among the highesthits.

High-ranking hits are those having a low E-value. The lower the E-value,the more significant the score (or in other words the lower the chancethat the hit was found by chance). Computation of the E-value is wellknown in the art. In addition to E-values, comparisons are also scoredby percentage identity. Percentage identity refers to the number ofidentical nucleotides (or amino acids) between the two compared nucleicacid (or polypeptide) sequences over a particular length. In the case oflarge families, ClustalW may be used, followed by a neighbour joiningtree, to help visualize clustering of related genes and to identifyorthologues and paralogues.

Table D gives examples of orthologues and paralogues of the MADS15protein represented by SEQ ID NO 44. Further orthologues and paraloguesmay readily be identified using the BLAST procedure described above.

The proteins of the invention are identifiable by the presence of theconserved MADS and/or keratin domain(s) (shown in FIG. 5). Specialistdatabases exist for the identification of domains, for example, SMART(Schultz et al. (1998) Proc. Natl. Acad. Sci. USA 95, 5857-5864; Letunicet al. (2002) Nucleic Acids Res 30, 242-244, InterPro (Mulder et al.,(2003) Nucl. Acids. Res. 31, 315-318, Prosite (Bucher and Bairoch(1994), A generalized profile syntax for biomolecular sequences motifsand its function in automatic sequence interpretation. (In) ISMB-94;Proceedings 2nd International Conference on Intelligent Systems forMolecular Biology. Altman R., Brutlag D., Karp P., Lathrop R., SearlsD., Eds., pp 53-61, AAAIPress, Menlo Park; Hulo et al., Nucl. Acids.Res. 32:D134-D137, (2004), or Pfam (Bateman et al., Nucleic AcidsResearch 30(1): 276-280 (2002). A set of tools for in silico analysis ofprotein sequences is available on the ExPASY proteomics server (hostedby the Swiss Institute of Bioinformatics (Gasteiger et al., ExPASy: theproteomics server for in-depth protein knowledge and analysis, NucleicAcids Res. 31:3784-3788 (2003)).

Domains may also be identified using routine techniques, such as bysequence alignment. Methods for the alignment of sequences forcomparison are well known in the art, such methods include GAP, BESTFIT,BLAST, FASTA and TFASTA. GAP uses the algorithm of Needleman and Wunsch((1970) J Mol Biol 48: 443-453) to find the global (i.e. spanning thecomplete sequences) alignment of two sequences that maximizes the numberof matches and minimizes the number of gaps. The BLAST algorithm(Altschul et al. (1990) J Mol Biol 215: 403-10) calculates percentsequence identity and performs a statistical analysis of the similaritybetween the two sequences. The software for performing BLAST analysis ispublicly available through the National Centre for BiotechnologyInformation (NCBI). Homologues may readily be identified using, forexample, the ClustalW multiple sequence alignment algorithm (version1.83), with the default pairwise alignment parameters, and a scoringmethod in percentage. Global percentages of similarity and identity mayalso be determined using one of the methods available in the MatGATsoftware package (Campanella et al., BMC Bioinformatics. 2003 Jul. 10;4:29. MatGAT: an application that generates similarity/identity matricesusing protein or DNA sequences.). Minor manual editing may be performedto optimise alignment between conserved motifs, as would be apparent toa person skilled in the art. Furthermore, instead of using full-lengthsequences for the identification of homologues, specific domains (suchas the MADS or keratin domain, or one of the motifs defined above) maybe used as well. The sequence identity values, which are indicated belowin Example 10 as a percentage were determined over the entire nucleicacid or amino acid sequence, and/or over selected domains or conservedmotif(s), using the programs mentioned above using the defaultparameters.

Furthermore, a MADS15 protein may also be identifiable by its ability orinability to bind DNA and to interact with other proteins. DNA-bindingactivity and protein-protein interactions may readily be determined invitro or in vivo using techniques well known in the art. Examples of invitro assays for DNA binding activity include: gel retardation analysisusing known MADS-box DNA binding domains (West et al. (1998) Nucl AcidRes 26(23): 5277-87), or yeast one-hybrid assays. An example of an invitro assay for protein-protein interactions is the yeast two-hybridanalysis (Fields and Song (1989) Nature 340:245-6). Proteins known tointeract with OsMADS15 include other MADS proteins (such as thoseinvolved in the flowering signalling complex), receptor like kinasessuch a Clavata1 and Clavata2, Erecta, BRI1 or RSK. Further details areprovided in Example 13.

Nucleic acids encoding proteins useful in the methods of the inventionneed not be full-length nucleic acids, since performance of the methodsof the invention does not rely on the use of full-length nucleic acidsequences. Examples of nucleic acids suitable for use in performing themethods of the invention include the nucleic acid sequences given intable D, but are not limited to those sequences. Nucleic acid variantsmay also be useful in practising the methods of the invention. Examplesof such nucleic acid variants include portions of nucleic acids encodinga protein useful in the methods of the invention, nucleic acidshybridising to nucleic acids encoding a protein useful in the methods ofthe invention, splice variants of nucleic acids encoding a proteinuseful in the methods of the invention, allelic variants of nucleicacids encoding a protein useful in the methods of the invention andvariants of nucleic acids encoding a protein useful in the methods ofthe invention that are obtained by gene shuffling. The terms portion,hybridising sequence, splice variant, allelic variant and gene shufflingwill now be described.

According to the present invention, there is provided a method forenhancing yield-related traits in plants, comprising introducing andexpressing in a plant a portion of any one of the nucleic acid sequencesgiven in table D, or a portion of a nucleic acid encoding an orthologue,paralogue or homologue of any of the amino acid sequences given in tableD.

Portions useful in the methods of the invention, encode a polypeptidefalling within the definition of a nucleic acid encoding a proteinuseful in the methods of the invention as defined herein and havingsubstantially the same biological activity as the amino acid sequencesgiven in table D. Preferably, the portion is a portion of any one of thenucleic acids given in table D. The portion is typically at least 400consecutive nucleotides in length, preferably at least 600 consecutivenucleotides in length, more preferably at least 700 consecutivenucleotides in length and most preferably at least 800 consecutivenucleotides in length, the consecutive nucleotides being of any one ofthe nucleic acid sequences given in table D. Most preferably the portionis a portion of the nucleic acid of SEQ ID NO: 43. Preferably, theportion encodes an amino acid sequence comprising (any one or more of)the MADS and keratin domain as defined herein.

A portion of a nucleic acid encoding a MADS15 protein as defined hereinmay be prepared, for example, by making one or more deletions to thenucleic acid. The portions may be used in isolated form or they may befused to other coding (or non coding) sequences in order to, forexample, produce a protein that combines several activities. When fusedto other coding sequences, the resultant polypeptide produced upontranslation may be bigger than that predicted for the MADS15 proteinportion.

Another nucleic acid variant useful in the methods of the invention is anucleic acid capable of hybridising, under reduced stringencyconditions, preferably under stringent conditions, with a nucleic acidencoding a MADS15 protein as defined herein, or with a portion asdefined herein.

Hybridising sequences useful in the methods of the invention, encode apolypeptide having a MADS and/or keratin domain (see the alignment ofFIG. 6) and having substantially the same biological activity as theMADS15 protein represented by any of the amino acid sequences given intable D. The hybridising sequence is typically at least 400 consecutivenucleotides in length, preferably at least 600 consecutive nucleotidesin length, more preferably at least 700 consecutive nucleotides inlength and most preferably at least 800 consecutive nucleotides inlength, the consecutive nucleotides being of any one of the nucleic acidsequences given in table D. Preferably, the hybridising sequence is onethat is capable of hybridising to any of the nucleic acids given intable D, or to a portion of any of these sequences, a portion being asdefined above. Most preferably, the hybridising sequence is capable ofhybridising to a nucleic acid as represented by SEQ ID NO: 43 or to aportion thereof. Preferably, the hybridising sequence encodes an aminoacid sequence comprising any one or more of the motifs or domains asdefined herein.

According to the present invention, there is provided a method forenhancing yield-related traits in plants, comprising introducing andexpressing in a plant a nucleic acid capable of hybridizing to any oneof the nucleic acids given in table D, or comprising introducing andexpressing in a plant a nucleic acid capable of hybridising to a nucleicacid encoding an orthologue, paralogue or homologue of any of thenucleic acid sequences given in table D.

Another nucleic acid variant useful in the methods of the invention is asplice variant encoding a MADS15 protein as defined hereinabove.

According to the present invention, there is provided a method forenhancing yield-related traits in plants, comprising introducing andexpressing in a plant a splice variant of any one of the nucleic acidsequences given in table D, or a splice variant of a nucleic acidencoding an orthologue, paralogue or homologue of any of the amino acidsequences given in table D.

Preferred splice variants are splice variants of a nucleic acidrepresented by SEQ ID NO: 43 or a splice variant of a nucleic acidencoding an orthologue or paralogue of SEQ ID NO: 44. Preferably, theamino acid sequence encoded by the splice variant comprises any one ormore of the motifs or domains as defined herein.

Another nucleic acid variant useful in performing the methods of theinvention is an allelic variant of a nucleic acid encoding a MADS15protein as defined hereinabove. The allelic variants useful in themethods of the present invention have substantially the same biologicalactivity as the MADS15 protein of SEQ ID NO: 44.

According to the present invention, there is provided a method forenhancing yield-related traits in plants, comprising introducing andexpressing in a plant an allelic variant of any one of the nucleic acidsgiven in table D, or comprising introducing and expressing in a plant anallelic variant of a nucleic acid encoding an orthologue, paralogue orhomologue of any of the amino acid sequences given in table D.

Preferably, the allelic variant is an allelic variant of SEQ ID NO: 43or an allelic variant of a nucleic acid encoding an orthologue orparalogue of SEQ ID NO: 44. Preferably, the amino acid sequence encodedby the allelic variant comprises any one or more of the motifs ordomains as defined herein.

A further nucleic acid variant useful in the methods of the invention isa nucleic acid variant obtained by gene shuffling. Gene shuffling ordirected evolution may also be used to generate variants of nucleicacids encoding MADS15 proteins as defined above.

According to the present invention, there is provided a method forenhancing yield-related traits in plants, comprising introducing andexpressing in a plant a variant of any one of the nucleic acid sequencesgiven in table D, or comprising introducing and expressing in a plant avariant of a nucleic acid encoding an orthologue, paralogue or homologueof any of the amino acid sequences given in table D, which variantnucleic acid is obtained by gene shuffling.

Preferably, the variant nucleic acid obtained by gene shuffling encodesan amino acid sequence comprising any one or more of the motifs ordomains as defined herein.

Furthermore, nucleic acid variants may also be obtained by site-directedmutagenesis.

Several methods are available to achieve site-directed mutagenesis, themost common being PCR based methods (Current Protocols in MolecularBiology. Wiley Eds.).

Nucleic acids encoding MADS15 proteins may be derived from any naturalor artificial source. The nucleic acid may be modified from its nativeform in composition and/or genomic environment through deliberate humanmanipulation. Preferably the MADS15-encoding nucleic acid is from aplant, further preferably from a monocotyledonous plant, more preferablyfrom the Poaceae family, most preferably the nucleic acid is from Oryzasativa.

Any reference herein to a MADS15 protein is therefore taken to mean aMADS15 protein as defined above. Any nucleic acid encoding such a MADS15protein is suitable for use in performing the methods of the invention.

The present invention also encompasses plants or parts thereof(including seeds) obtainable by the methods according to the presentinvention. The plants or parts thereof comprise a nucleic acid transgeneencoding a MADS15 protein as defined above.

The invention also provides genetic constructs and vectors to facilitateintroduction and/or expression of the nucleic acid sequences useful inthe methods according to the invention, in a plant. The gene constructsmay be inserted into vectors, which may be commercially available,suitable for transforming into plants and suitable for expression of thegene of interest in the transformed cells. The invention also providesuse of a gene construct as defined herein in the methods of theinvention.

More specifically, the present invention provides a construct comprising

-   -   (a) nucleic acid encoding MADS15 protein as defined above;    -   (b) one or more control sequences capable of driving expression        of the nucleic acid sequence of (a); and optionally    -   (c) a transcription termination sequence.

Plants are transformed with a vector comprising the sequence of interest(i.e., a nucleic acid encoding a MADS15 polypeptide as defined herein.The skilled artisan is well aware of the genetic elements that must bepresent on the vector in order to successfully transform, select andpropagate host cells containing the sequence of interest. The sequenceof interest is operably linked to one or more control sequences (atleast to a promoter).

Advantageously, any type of promoter may be used to drive expression ofthe nucleic acid sequence.

The promoter may be a constitutive promoter; alternatively, the promotermay be an inducible promoter, i.e. having induced or increasedtranscription initiation in response to a chemical or a stress-induciblepromoter, or a pathogen-induced promoter.

Additionally or alternatively, the promoter may be an organ-specific ortissue-specific promoter, or the promoter may be a ubiquitous promoter,or the promoter may be developmentally regulated. Furthermore, thepromoter may be organ-specific or tissue-specific or cell-specific.

Preferably, the MADS15 nucleic acid or variant thereof is operablylinked to a constitutive promoter. A preferred constitutive promoter isone that is also substantially ubiquitously expressed. Furtherpreferably the promoter is derived from a plant, more preferably amonocotyledonous plant. Most preferred is use of a GOS2 promoter (forexample from rice, SEQ ID NO: 47 or SEQ ID NO: 108). It should be clearthat the applicability of the present invention is not restricted to theMADS15 nucleic acid represented by SEQ ID NO: 43, nor is theapplicability of the invention restricted to expression of a MADS15nucleic acid when driven by a GOS2 promoter. Examples of otherconstitutive promoters or functionally equivalent promoters which mayalso be used to drive expression of a MADS15 nucleic acid are shown inthe definitions section.

Optionally, one or more terminator sequences may be used in theconstruct introduced into a plant. Additional regulatory elements mayinclude transcriptional as well as translational enhancers. Thoseskilled in the art will be aware of terminator and enhancer sequencesthat may be suitable for use in performing the invention. Such sequenceswould be known or may readily be obtained by a person skilled in theart.

An intron sequence may also be added to the 5′ untranslated region (UTR)or in the coding sequence to increase the amount of the mature messagethat accumulates in the cytosol.

Other control sequences (besides promoter, enhancer, silencer, intronsequences, 3′UTR and/or 5′UTR regions) may be protein and/or RNAstabilizing elements. Such sequences would be known or may readily beobtained by a person skilled in the art.

The genetic constructs of the invention may further include an origin ofreplication sequence that is required for maintenance and/or replicationin a specific cell type. One example is when a genetic construct isrequired to be maintained in a bacterial cell as an episomal geneticelement (e.g. plasmid or cosmid molecule). Preferred origins ofreplication include, but are not limited to, the f1-ori and colE1.

For the detection of the successful transfer of the nucleic acidsequences as used in the methods of the invention and/or selection oftransgenic plants comprising these nucleic acids, it is advantageous touse marker genes (or reporter genes). Therefore, the genetic constructmay optionally comprise a selectable marker gene.

The invention also provides a method for the production of transgenicplants having enhanced yield-related traits relative to control plants,comprising introduction and expression in a plant of any nucleic acidencoding a MADS15 protein as defined hereinabove.

More specifically, the present invention provides a method for theproduction of transgenic plants having increased yield, which methodcomprises:

-   -   (i) introducing and expressing in a plant or plant cell a MADS15        nucleic acid or variant thereof; and    -   (ii) cultivating the plant cell under conditions promoting plant        growth and development.

The nucleic acid may be introduced directly into a plant cell or intothe plant itself (including introduction into a tissue, organ or anyother part of a plant). According to a preferred feature of the presentinvention, the nucleic acid is preferably introduced into a plant bytransformation.

The genetically modified plant cells can be regenerated via all methodswith which the skilled worker is familiar. Suitable methods can be foundin the abovementioned publications by S. D. Kung and R. Wu, Potrykus orHöfgen and Willmitzer.

Generally after transformation, plant cells or cell groupings areselected for the presence of one or more markers which are encoded byplant-expressible genes co-transferred with the gene of interest,following which the transformed material is regenerated into a wholeplant. To select transformed plants, the plant material obtained in thetransformation is, as a rule, subjected to selective conditions so thattransformed plants can be distinguished from untransformed plants. Forexample, the seeds obtained in the above-described manner can be plantedand, after an initial growing period, subjected to a suitable selectionby spraying. A further possibility consists in growing the seeds, ifappropriate after sterilization, on agar plates using a suitableselection agent so that only the transformed seeds can grow into plants.Alternatively, the transformed plants are screened for the presence of aselectable marker such as the ones described above.

Following DNA transfer and regeneration, putatively transformed plantsmay also be evaluated, for instance using Southern analysis, for thepresence of the gene of interest, copy number and/or genomicorganisation. Alternatively or additionally, expression levels of thenewly introduced DNA may be monitored using Northern and/or Westernanalysis, both techniques being well known to persons having ordinaryskill in the art.

The generated transformed plants may be propagated by a variety ofmeans, such as by clonal propagation or classical breeding techniques.For example, a first generation (or T1) transformed plant may be selfedand homozygous second-generation (or T2) transformants selected, and theT2 plants may then further be propagated through classical breedingtechniques.

The generated transformed organisms may take a variety of forms. Forexample, they may be chimeras of transformed cells and non-transformedcells; clonal transformants (e.g., all cells transformed to contain theexpression cassette); grafts of transformed and untransformed tissues(e.g., in plants, a transformed rootstock grafted to an untransformedscion).

The present invention clearly extends to any plant cell or plantproduced by any of the methods described herein, and to all plant partsand propagules thereof. The present invention extends further toencompass the progeny of a primary transformed or transfected cell,tissue, organ or whole plant that has been produced by any of theaforementioned methods, the only requirement being that progeny exhibitthe same genotypic and/or phenotypic characteristic(s) as those producedby the parent in the methods according to the invention.

The invention also includes host cells containing an isolated nucleicacid encoding a MADS15 protein as defined hereinabove. Preferred hostcells according to the invention are plant cells.

Host plants for the nucleic acids or the vector used in the methodaccording to the invention, the expression cassette or construct orvector are, in principle, advantageously all plants, which are capableof synthesizing the polypeptides used in the inventive method.

The invention also extends to harvestable parts of a plant such as, butnot limited to seeds, leaves, fruits, flowers, stems, rhizomes, roots,tubers and bulbs. The invention furthermore relates to products derived,preferably directly derived, from a harvestable part of such a plant,such as dry pellets or powders, oil, fat and fatty acids, starch orproteins.

According to a preferred feature of the invention, the modulatedexpression is increased expression.

As mentioned above, a preferred method for modulating (preferably,increasing) expression of a nucleic acid encoding a MADS15 protein is byintroducing and expressing in a plant a nucleic acid encoding a MADS15protein; however the effects of performing the method, i.e. enhancingyield-related traits may also be achieved using other well knowntechniques. A description of some of these techniques will now follow.

One such technique is T-DNA activation tagging. The resulting transgenicplants show dominant phenotypes due to modified expression of genesclose to the introduced promoter.

The effects of the invention may also be reproduced using the techniqueof TILLING (Targeted Induced Local Lesions In Genomes), or by homologousrecombination.

Reference herein to enhanced yield-related traits is taken to mean anincrease in biomass (weight) of one or more parts of a plant, which mayinclude aboveground (harvestable) parts and/or (harvestable) parts belowground.

Taking corn as an example, a yield increase may be manifested as one ormore of the following: increase in the number of plants established perhectare or acre, an increase in the number of ears per plant, anincrease in the number of rows, number of kernels per row, kernelweight, thousand kernel weight, ear length/diameter, increase in theseed filling rate (which is the number of filled seeds divided by thetotal number of seeds and multiplied by 100), among others. Taking riceas an example, a yield increase may manifest itself as an increase inone or more of the following: number of plants per hectare or acre,number of panicles per plant, number of spikelets per panicle, number offlowers (florets) per panicle (which is expressed as a ratio of thenumber of filled seeds over the number of primary panicles), increase inthe seed filling rate (which is the number of filled seeds divided bythe total number of seeds and multiplied by 100), increase in thousandkernel weight, among others.

In a particular embodiment, such harvestable parts are roots, andperformance of the methods of the invention results in plants havingincreased root growth relative to the root growth of suitable controlplants. Root development is an essential determinant of plant growth andcrop yield since the root is the main channel to extract nutrients fromthe environment.

Increased root yield may for example manifest itself as one of more ofthe following: a) increased amount of root biomass (whereby adiscrimination between thick roots and thin roots may be made bydefining a certain threshold), b) increased average root diameter, c)increased total root biomass, and d) increased root biomass/shootbiomass ratio. For the purpose of this invention, it should beunderstood that the term ‘root growth’ encompasses all aspects of growthof the different parts that make up the root system at different stagesof its development, both in monocotyledonous and dicotyledonous plants.It is to be understood that enhanced growth of the root can result fromenhanced growth of one or more of its parts including the primary root,lateral roots, adventitious roots, etc. all of which fall within thescope of this invention.

Since the transgenic plants according to the present invention haveincreased yield, it is likely that these plants exhibit an increasedgrowth rate (during at least part of their life cycle), relative to thegrowth rate of control plants at a corresponding stage in their lifecycle. The increased growth rate may be specific to one or more parts ofa plant (including seeds), or may be throughout substantially the wholeplant. Plants having an increased growth rate may have a shorter lifecycle. The life cycle of a plant may be taken to mean the time needed togrow from a dry mature seed up to the stage where the plant has produceddry mature seeds, similar to the starting material. This life cycle maybe influenced by factors such as early vigour, growth rate, greennessindex, flowering time and speed of seed maturation. The increase ingrowth rate may take place at one or more stages in the life cycle of aplant or during substantially the whole plant life cycle. Increasedgrowth rate during the early stages in the life cycle of a plant mayreflect enhanced vigour. The increase in growth rate may alter theharvest cycle of a plant allowing plants to be sown later and/orharvested sooner than would otherwise be possible (a similar effect maybe obtained with earlier flowering time). If the growth rate issufficiently increased, it may allow for the further sowing of seeds ofthe same plant species (for example sowing and harvesting of rice plantsfollowed by sowing and harvesting of further rice plants all within oneconventional growing period). Similarly, if the growth rate issufficiently increased, it may allow for the further sowing of seeds ofdifferent plants species (for example the sowing and harvesting of cornplants followed by, for example, the sowing and optional harvesting ofsoy bean, potato or any other suitable plant). Harvesting additionaltimes from the same rootstock in the case of some crop plants may alsobe possible. Altering the harvest cycle of a plant may lead to anincrease in annual biomass production per acre (due to an increase inthe number of times (say in a year) that any particular plant may begrown and harvested). An increase in growth rate may also allow for thecultivation of transgenic plants in a wider geographical area than theirwild-type counterparts, since the territorial limitations for growing acrop are often determined by adverse environmental conditions either atthe time of planting (early season) or at the time of harvesting (lateseason). Such adverse conditions may be avoided if the harvest cycle isshortened. The growth rate may be determined by deriving variousparameters from growth curves, such parameters may be: T-Mid (the timetaken for plants to reach 50% of their maximal size) and T-90 (timetaken for plants to reach 90% of their maximal size), amongst others.

According to a preferred feature of the present invention, performanceof the methods of the invention gives plants having an increased growthrate relative to control plants. Therefore, according to the presentinvention, there is provided a method for increasing the growth rate ofplants, which method comprises modulating expression, preferablyincreasing expression, in a plant of a nucleic acid encoding a MADS15protein as defined herein.

An increase in yield and/or growth rate occurs whether the plant isunder non-stress conditions or whether the plant is exposed to variousstresses compared to control plants. Plants typically respond toexposure to stress by growing more slowly. In conditions of severestress, the plant may even stop growing altogether. Mild stress on theother hand is defined herein as being any stress to which a plant isexposed which does not result in the plant ceasing to grow altogetherwithout the capacity to resume growth. Mild stress in the sense of theinvention leads to a reduction in the growth of the stressed plants ofless than 40%, 35% or 30%, preferably less than 25%, 20% or 15%, morepreferably less than 14%, 13%, 12%, 11% or 10% or less in comparison tothe control plant under non-stress conditions. Due to advances inagricultural practices (irrigation, fertilization, pesticide treatments)severe stresses are not often encountered in cultivated crop plants. Asa consequence, the compromised growth induced by mild stress is often anundesirable feature for agriculture. Mild stresses are the everydaybiotic and/or abiotic (environmental) stresses to which a plant isexposed. Abiotic stresses may be due to drought or excess water,anaerobic stress, salt stress, chemical toxicity, oxidative stress andhot, cold or freezing temperatures. The abiotic stress may be an osmoticstress caused by a water stress (particularly due to drought), saltstress, oxidative stress or an ionic stress. Biotic stresses aretypically those stresses caused by pathogens, such as bacteria, viruses,fungi and insects.

In particular, the methods of the present invention may be performedunder non-stress conditions or under conditions of mild drought to giveplants having increased yield relative to control plants. As reported inWang et al. (Planta (2003) 218: 1-14), abiotic stress leads to a seriesof morphological, physiological, biochemical and molecular changes thatadversely affect plant growth and productivity. Drought, salinity,extreme temperatures and oxidative stress are known to be interconnectedand may induce growth and cellular damage through similar mechanisms.Rabbani et al. (Plant Physiol (2003) 133: 1755-1767) describes aparticularly high degree of “cross talk” between drought stress andhigh-salinity stress. For example, drought and/or salinisation aremanifested primarily as osmotic stress, resulting in the disruption ofhomeostasis and ion distribution in the cell. Oxidative stress, whichfrequently accompanies high or low temperature, salinity or droughtstress, may cause denaturing of functional and structural proteins. As aconsequence, these diverse environmental stresses often activate similarcell signaling pathways and cellular responses, such as the productionof stress proteins, up-regulation of anti-oxidants, accumulation ofcompatible solutes and growth arrest. The term “non-stress” conditionsas used herein are those environmental conditions that allow optimalgrowth of plants. Persons skilled in the art are aware of normal soilconditions and climatic conditions for a given location.

Performance of the methods of the invention gives plants grown undernon-stress conditions or under mild drought conditions increased yieldrelative to control plants grown under comparable conditions. Therefore,according to the present invention, there is provided a method forincreasing yield in plants grown under non-stress conditions or undermild drought conditions, which method comprises increasing expression ina plant of a nucleic acid encoding a MADS15 polypeptide.

Performance of the methods of the invention gives plants grown underconditions of nutrient deficiency, particularly under conditions ofnitrogen deficiency, increased yield relative to control plants grownunder comparable conditions. Therefore, according to the presentinvention, there is provided a method for increasing yield in plantsgrown under conditions of nutrient deficiency, which method comprisesincreasing expression in a plant of a nucleic acid encoding a MADS15polypeptide. Nutrient deficiency may result from a lack or excess ofnutrients such as nitrogen, phosphates and other phosphorous-containingcompounds, potassium, calcium, cadmium, magnesium, manganese, iron andboron, amongst others.

In a preferred embodiment of the invention, the increase in yield and/orgrowth rate occurs according to the methods of the present inventionunder non-stress conditions.

The methods of the invention are advantageously applicable to any plant.

Plants that are particularly useful in the methods of the inventioninclude all plants which belong to the superfamily Viridiplantae, inparticular monocotyledonous and dicotyledonous plants including fodderor forage legumes, ornamental plants, food crops, trees or shrubs.According to a preferred embodiment of the present invention, the plantis a crop plant. Examples of crop plants include soybean, sunflower,canola, alfalfa, rapeseed, cotton, tomato, potato and tobacco. Furtherpreferably, the plant is a monocotyledonous plant. Examples ofmonocotyledonous plants include sugarcane. More preferably the plant isa cereal. Examples of cereals include rice, maize, wheat, barley,millet, rye, triticale, sorghum and oats.

The present invention also encompasses use of nucleic acids encoding theMADS15 protein described herein and use of these MADS15 proteins inenhancing yield-related traits in plants.

Nucleic acids encoding the MADS15 protein described herein, or theMADS15 proteins themselves, may find use in breeding programmes in whicha DNA marker is identified which may be genetically linked to aMADS15-encoding gene. The nucleic acids/genes, or the MADS15 proteinsthemselves may be used to define a molecular marker. This DNA or proteinmarker may then be used in breeding programmes to select plants havingenhanced yield-related traits as defined hereinabove in the methods ofthe invention.

Allelic variants of a MADS15 protein-encoding nucleic acid/gene may alsofind use in marker-assisted breeding programmes. Such breedingprogrammes sometimes require introduction of allelic variation bymutagenic treatment of the plants, using for example EMS mutagenesis;alternatively, the programme may start with a collection of allelicvariants of so called “natural” origin caused unintentionally.Identification of allelic variants then takes place, for example, byPCR. This is followed by a step for selection of superior allelicvariants of the sequence in question and which give increased yield.Selection is typically carried out by monitoring growth performance ofplants containing different allelic variants of the sequence inquestion. Growth performance may be monitored in a greenhouse or in thefield. Further optional steps include crossing plants in which thesuperior allelic variant was identified with another plant. This couldbe used, for example, to make a combination of interesting phenotypicfeatures.

Nucleic acids encoding MADS15 proteins may also be used as probes forgenetically and physically mapping the genes that they are a part of,and as markers for traits linked to those genes. Such information may beuseful in plant breeding in order to develop lines with desiredphenotypes. Such use of MADS15 protein-encoding nucleic acids requiresonly a nucleic acid sequence of at least 15 nucleotides in length. TheMADS15 protein-encoding nucleic acids may be used as restrictionfragment length polymorphism (RFLP) markers. Southern blots (Sambrook J,Fritsch E F and Maniatis T (1989) Molecular Cloning, A LaboratoryManual) of restriction-digested plant genomic DNA may be probed with theMADS15 protein-encoding nucleic acids. The resulting banding patternsmay then be subjected to genetic analyses using computer programs suchas MapMaker (Lander et al. (1987) Genomics 1: 174-181) in order toconstruct a genetic map. In addition, the nucleic acids may be used toprobe Southern blots containing restriction endonuclease-treated genomicDNAs of a set of individuals representing parent and progeny of adefined genetic cross. Segregation of the DNA polymorphisms is noted andused to calculate the position of the MADS15 protein-encoding nucleicacid in the genetic map previously obtained using this population(Botstein et al. (1980) Am. J. Hum. Genet. 32:314-331).

The production and use of plant gene-derived probes for use in geneticmapping is described in Bernatzky and Tanksley (1986) Plant Mol. Biol.Reporter 4: 37-41. Numerous publications describe genetic mapping ofspecific cDNA clones using the methodology outlined above or variationsthereof. For example, F2 intercross populations, backcross populations,randomly mated populations, near isogenic lines, and other sets ofindividuals may be used for mapping. Such methodologies are well knownto those skilled in the art.

The nucleic acid probes may also be used for physical mapping (i.e.,placement of sequences on physical maps; see Hoheisel et al. In:Non-mammalian Genomic Analysis: A Practical Guide, Academic press 1996,pp. 319-346, and references cited therein).

In another embodiment, the nucleic acid probes may be used in directfluorescence in situ hybridisation (FISH) mapping (Trask (1991) TrendsGenet. 7:149-154). Although current methods of FISH mapping favour useof large clones (several kb to several hundred kb; see Laan et al.(1995) Genome Res. 5:13-20), improvements in sensitivity may allowperformance of FISH mapping using shorter probes.

A variety of nucleic acid amplification-based methods for genetic andphysical mapping may be carried out using the nucleic acids. Examplesinclude allele-specific amplification (Kazazian (1989) J. Lab. Clin. Med11:95-96), polymorphism of PCR-amplified fragments (CAPS; Sheffield etal. (1993) Genomics 16:325-332), allele-specific ligation (Landegren etal. (1988) Science 241:1077-1080), nucleotide extension reactions(Sokolov (1990) Nucleic Acid Res. 18:3671), Radiation Hybrid Mapping(Walter et al. (1997) Nat. Genet. 7:22-28) and Happy Mapping (Dear andCook (1989) Nucleic Acid Res. 17:6795-6807). For these methods, thesequence of a nucleic acid is used to design and produce primer pairsfor use in the amplification reaction or in primer extension reactions.The design of such primers is well known to those skilled in the art. Inmethods employing PCR-based genetic mapping, it may be necessary toidentify DNA sequence differences between the parents of the mappingcross in the region corresponding to the instant nucleic acid sequence.This, however, is generally not necessary for mapping methods.

The methods according to the present invention result in plants havingenhanced yield-related traits, as described hereinbefore. These traitsmay also be combined with other economically advantageous traits, suchas further yield-enhancing traits, tolerance to other abiotic and bioticstresses, traits modifying various architectural features and/orbiochemical and/or physiological features.

Detailed Description of MADS15 Down

It has now surprisingly been found that decreasing the level and/oractivity of an endogenous MADS15 polypeptide gives plants havingenhanced yield-related traits, in particular increased yield relative,to corresponding wild type plants. The present invention thereforeprovides methods for enhancing yield related traits, particularly forincreasing yield of a plant relative to control plants, comprisingdecreasing the level and/or activity of an endogenous MADS15polypeptide. Reference herein to “control plants” is taken to meancorresponding wild type plants in which there is no reduction inactivity of the endogenous MADS15 polypeptide.

Advantageously, performance of the methods according to the presentinvention results in plants having increased yield, particularlyincreased seed yield and/or increased biomass, relative to correspondingwild type plants.

The term “increased yield” as defined herein is taken to mean anincrease in biomass (weight) of one or more parts of a plant, which mayinclude aboveground (harvestable) parts and/or (harvestable) parts belowground.

In particular, such harvestable parts include vegetative biomass and/orseeds, and performance of the methods of the invention results in plantshaving increased yield (in vegetative biomass and/or seed) relative tothe yield of control plants.

Taking corn as an example, a yield increase may be manifested as one ormore of the following: increase in the number of plants per hectare oracre, an increase in the number of ears per plant, an increase in thenumber of rows, number of kernels per row, kernel weight, thousandkernel weight, ear length/diameter, increase in the seed filling rate(which is the number of filled seeds divided by the total number ofseeds and multiplied by 100), among others. Taking rice as an example, ayield increase may manifest itself as an increase in one or more of thefollowing: number of plants per hectare or acre, number of panicles perplant, number of spikelets per panicle, number of flowers (florets) perpanicle (which is expressed as a ratio of the number of filled seedsover the number of primary panicles), increase in the seed filling rate(which is the number of filled seeds divided by the total number ofseeds and multiplied by 100), increase in thousand kernel weight, amongothers.

The increase in seed yield may also be manifested as an increase in seedsize and/or seed volume. This may increase the amount, or change thecomposition of, substances in the seed, such as oils, proteins andcarbohydrates.

According to a preferred feature, performance of the methods of theinvention result in plants having increased yield, particularlyincreased biomass and/or seed yield. Therefore, according to the presentinvention, there is provided a method for increasing plant yield, whichmethod comprises decreasing the level of activity of a MADS15polypeptide or a homologue thereof, preferably by downregulatingexpression of a MADS15 gene or a homologue thereof.

Since the transgenic plants according to the present invention haveincreased yield, it is likely that these plants exhibit an increasedgrowth rate (during at least part of their life cycle), relative to thegrowth rate of corresponding wild type plants at a corresponding stagein their life cycle. The increased growth rate may be specific to one ormore parts of a plant (including seeds), or may be throughoutsubstantially the whole plant. Plants having an increased growth ratemay have a shorter life cycle. The life cycle of a plant may be taken tomean the time needed to grow from a dry mature seed up to the stagewhere the plant has produced dry mature seeds, similar to the startingmaterial. This life cycle may be influenced by factors such as earlyvigour, growth rate, flowering time and speed of seed maturation. Anincrease in growth rate may take place at one or more stages in the lifecycle of a plant or during substantially the whole plant life cycle.Increased growth rate during the early stages in the life cycle of aplant may reflect enhanced vigour. The increase in growth rate may alterthe harvest cycle of a plant allowing plants to be sown later and/orharvested sooner than would otherwise be possible (a similar effect maybe obtained with earlier flowering time). If the growth rate issufficiently increased, it may allow for the further sowing of seeds ofthe same plant species (for example sowing and harvesting of rice plantsfollowed by sowing and harvesting of further rice plants all within oneconventional growing period). Similarly, if the growth rate issufficiently increased, it may allow for the further sowing of seeds ofdifferent plants species (for example the sowing and harvesting of cornplants followed by, for example, the sowing and optional harvesting ofsoy bean, potato or any other suitable plant). Harvesting additionaltimes from the same rootstock in the case of some crop plants may alsobe possible. Altering the harvest cycle of a plant may lead to anincrease in annual biomass production per acre (due to an increase inthe number of times (say in a year) that any particular plant may begrown and harvested). An increase in growth rate may also allow for thecultivation of transgenic plants in a wider geographical area than theirwild-type counterparts, since the territorial limitations for growing acrop are often determined by adverse environmental conditions either atthe time of planting (early season) or at the time of harvesting (lateseason). Such adverse conditions may be avoided if the harvest cycle isshortened. The growth rate may be determined by deriving variousparameters from growth curves, such parameters may be: T-Mid (the timetaken for plants to reach 50% of their maximal size) and T-90 (timetaken for plants to reach 90% of their maximal size), amongst others.

Performance of the methods of the invention gives plants having anincreased growth rate. Therefore, according to the present invention,there is provided a method for increasing the growth rate of plantsrelative to control plants, which method comprises preferentiallyreducing the expression level and/or activity of an endogenous MADS15gene in a plant.

An increase in yield and/or growth rate occurs whether the plant isunder non-stress conditions or whether the plant is exposed to variousstresses compared to control plants. Plants typically respond toexposure to stress by growing more slowly. In conditions of severestress, the plant may even stop growing altogether. Mild stress on theother hand is defined herein as being any stress to which a plant isexposed which does not result in the plant ceasing to grow altogetherwithout the capacity to resume growth. Mild stress in the sense of theinvention leads to a reduction in the growth of the stressed plants ofless than 40%, 35% or 30%, preferably less than 25%, 20% or 15%, morepreferably less than 14%, 13%, 12%, 11% or 10% or less in comparison tothe control plant under non-stress conditions. Due to advances inagricultural practices (irrigation, fertilization, pesticide treatments)severe stresses are not often encountered in cultivated crop plants. Asa consequence, the compromised growth induced by mild stress is often anundesirable feature for agriculture. Mild stresses are the everydaybiotic and/or abiotic (environmental) stresses to which a plant isexposed. Abiotic stresses may be due to drought or excess water,anaerobic stress, salt stress, chemical toxicity, oxidative stress andhot, cold or freezing temperatures. The abiotic stress may be an osmoticstress caused by a water stress (particularly due to drought), saltstress, oxidative stress or an ionic stress. Biotic stresses aretypically those stresses caused by pathogens, such as bacteria, viruses,fungi and insects.

In particular, the methods of the present invention may be performedunder non-stress conditions or under conditions of mild drought to giveplants having increased yield relative to control plants. As reported inWang et al. (Planta (2003) 218: 1-14), abiotic stress leads to a seriesof morphological, physiological, biochemical and molecular changes thatadversely affect plant growth and productivity. Drought, salinity,extreme temperatures and oxidative stress are known to be interconnectedand may induce growth and cellular damage through similar mechanisms.Rabbani et al. (Plant Physiol (2003) 133: 1755-1767) describes aparticularly high degree of “cross talk” between drought stress andhigh-salinity stress. For example, drought and/or salinisation aremanifested primarily as osmotic stress, resulting in the disruption ofhomeostasis and ion distribution in the cell. Oxidative stress, whichfrequently accompanies high or low temperature, salinity or droughtstress, may cause denaturing of functional and structural proteins. As aconsequence, these diverse environmental stresses often activate similarcell signaling pathways and cellular responses, such as the productionof stress proteins, up-regulation of anti-oxidants, accumulation ofcompatible solutes and growth arrest. The term “non-stress” conditionsas used herein are those environmental conditions that allow optimalgrowth of plants. Persons skilled in the art are aware of normal soilconditions and climatic conditions for a given location.

Performance of the methods of the invention gives plants grown undernon-stress conditions or under mild drought conditions increased yieldrelative to control plants grown under comparable conditions. Therefore,according to the present invention, there is provided a method forincreasing yield in plants grown under non-stress conditions or undermild drought conditions, which method comprises reducing expression ofan endogenous MADS15 gene in a plant.

Performance of the methods of the invention gives plants grown underconditions of nutrient deficiency, particularly under conditions ofnitrogen deficiency, increased yield relative to control plants grownunder comparable conditions. Therefore, according to the presentinvention, there is provided a method for increasing yield in plantsgrown under conditions of nutrient deficiency, which method comprisesdecreasing expression in a plant of a nucleic acid encoding a MADS15polypeptide. Nutrient deficiency may result from a lack or excess ofnutrients such as nitrogen, phosphates and other phosphorous-containingcompounds, potassium, calcium, cadmium, magnesium, manganese, iron andboron, amongst others.

In a preferred embodiment of the invention, the increase in yield and/orgrowth rate occurs according to the methods of the present inventionunder non-stress conditions.

The methods of the invention are advantageously applicable to any plant.

Plants that are particularly useful in the methods of the inventioninclude all plants which belong to the superfamily Viridiplantae, inparticular monocotyledonous and dicotyledonous plants including fodderor forage legumes, ornamental plants, food crops, trees or shrubs.According to a preferred embodiment of the present invention, the plantis a crop plant. Examples of crop plants include soybean, sunflower,canola, alfalfa, rapeseed, cotton, tomato, potato and tobacco. Furtherpreferably, the plant is a monocotyledonous plant. Examples ofmonocotyledonous plants include sugarcane. More preferably the plant isa cereal. Examples of cereals include rice, maize, wheat, barley,millet, rye, triticale, sorghum and oats.

Reference herein to a “decrease” in level of an endogenous MADS15protein in a plant is taken to mean a reduction in protein concentrationor substantial elimination of an endogenous MADS15 protein relative toendogenous MADS15 protein levels found in corresponding wild typeplants. This reduction or substantial elimination may result in reducedor substantially abolished MADS15 protein activity in a plant.

Reference herein to a “decrease” in activity of an endogenous MADS15protein in a plant is taken to mean a reduction in MADS15 proteinactivity or substantial elimination of activity of an endogenous MADS15protein relative to endogenous MADS15 protein activity levels found wildtype plants.

Preferably, the reduction in endogenous MADS15 protein level and/oractivity is obtained by downregulating the expression of the endogenousMADS15 gene.

Reference herein to an “endogenous” MADS15 gene not only refers toMADS15 genes as found in a plant in its natural form (i.e., withoutthere being any human intervention), but also refers to isolated MADS15genes subsequently introduced into a plant. For example, a transgenicplant containing a MADS15 transgene may encounter a reduction orsubstantial elimination of the MADS15 transgene expression and/orreduced or substantial elimination of expression of an endogenous MADS15gene.

This reduction (or substantial elimination) of endogenous MADS15 geneexpression may be achieved using any one or more of several well-knowngene silencing methods. “Gene silencing” or “downregulation” ofexpression, as used herein, refers to a reduction or the substantialelimination of MADS15 gene expression and/or MADS15 polypeptide levelsand/or MADS15 polypeptide activity.

One such method for reduction or substantial elimination of endogenousMADS15 gene expression is RNA-mediated downregulation of gene expression(RNA silencing). Silencing in this case is triggered in a plant by adouble stranded RNA molecule (dsRNA) that is substantially homologous toa target MADS15 gene. This dsRNA is further processed by the plant intoabout 21 to about 26 nucleotides called short interfering RNAs (siRNAs).The siRNAs are incorporated into an RNA-induced silencing complex (RISC)that cleaves the mRNA of a MADS15 target gene, thereby reducing orsubstantially eliminating the number of MADS15 mRNAs to be translatedinto a MADS15 protein.

One example of an RNA silencing method involves the introduction ofcoding sequences or parts thereof in a sense orientation into a plant.The additional gene, or part thereof, will silence an endogenous MADS15gene, giving rise to a phenomenon known as co-suppression. The reductionof MADS15 gene expression will be more pronounced if several additionalcopies are introduced into the plant, as there is a positive correlationbetween high transcript levels and the triggering of co-suppression.

Another example of an RNA silencing method involves the use of antisenseMADS15 nucleic acid sequences.

The antisense nucleic acid can be produced biologically using anexpression vector into which a nucleic acid has been subcloned in anantisense orientation (i.e., RNA transcribed from the inserted nucleicacid will be of an antisense orientation to a target nucleic acid ofinterest, described further in the following subsection). Preferably,production of antisense nucleic acids in plants occurs by means of astably integrated transgene comprising a promoter operative forconstitutive expression in plants, an antisense oligonucleotide, and aterminator.

A preferred method for reduction or substantial elimination ofendogenous MADS15 gene expression via RNA silencing is by using anexpression vector into which a MADS15 gene or fragment thereof has beencloned as an inverted repeat (in part or completely), separated by aspacer (non-coding DNA).

In still another embodiment, the reduction or substantial elimination ofendogenous MADS15 expression may be obtained by using ribozymes. Forexample, a derivative of a Tetrahymena L-19 IVS RNA can be constructedin which the nucleotide sequence of the active site is complementary tothe nucleotide sequence to be cleaved in an MADS15-encoding mRNA.

Gene silencing may also be achieved by insertion mutagenesis or if thereis a mutation on the endogenous MADS15 gene and/or a mutation on anisolated MADS15 gene subsequently introduced into a plant. The reductionor substantial elimination of MADS15 protein activity may be caused by anon-functional MADS15. For example, MADS15 binds to various interactingproteins; one or more mutation(s) and/or truncation(s) within the MADSbox of a MADS15 may therefore provide for a MADS15 protein that is stillable to bind interacting proteins but that cannot exhibit its normalfunction as transcription factor.

A further approach to gene silencing is by targeting nucleotidesequences complementary to the regulatory region of the MADS15 gene(e.g., the MADS15 promoter and/or enhancers) to form triple helicalstructures that prevent transcription of the MADS15 gene in targetcells.

Still another approach to gene silencing is described by Hiratsu et al.(Plant J. 34, 733-739, 2003). This method does not depend on sequencehomology to the targeted gene but involves the use of a repressionsequence domain in transcriptional gene fusions, and has been used tomodify traits of agronomic interest (Fujita et al., Plant Cell 17,3470-3488, 2005 and Mitsuda at al., Plant Cell 17, 2993-3006, 2005).Typically, a nucleotide chimeric fusion is made between a gene encodinga protein capable of positively influencing the expression of thetargeted gene (such as a transcription activator), and a nucleotidefragment encoding a repression domain. Upon expression of the chimericgene fusion, the expression of the targeted gene is repressed, usuallyin a dominant negative fashion. Repression domains are well known in theart, for example the EAR motif present in some AP2 and Zinc fingertranscription factor. Methods based on repression domains are wellsuited to overcome gene redundancy for the targeted gene in the plantspecies of choice.

Described above are examples of various methods for gene silencing (forthe reduction or substantial elimination of endogenous MADS15 geneexpression. The methods of the invention rely on the reduction ofexpression of an endogenous MADS15 gene in a plant. A person skilled inthe art would readily be able to adapt the aforementioned methods forsilencing so as to achieve gene silencing in a whole plant or in partsthereof through the use of an appropriate promoter, for example.

It should be noted that the essence of the present invention resides inthe advantageous and surprising results found upon reduction orsubstantial elimination of endogenous MADS15 gene expression in a plant,and is not limited to any particular method for such reduction orsubstantial elimination of endogenous MADS15 protein activity. Theactivity of a MADS15 polypeptide may also be decreased or eliminated byintroducing a genetic modification (preferably in the locus of a MADS15gene). The locus of a gene as defined herein is taken to mean a genomicregion, which includes the gene of interest and 10 kb up- or down streamof the coding region.

The genetic modification may be introduced, for example, by any one (ormore) of the following methods: T-DNA inactivation, TILLING,site-directed mutagenesis, directed evolution, homologous recombination.Following introduction of the genetic modification, there follows a stepof selecting for decreased activity of a MADS15 polypeptide, whichdecrease in activity gives plants having increased yield.

T-DNA inactivation tagging involves insertion of a T-DNA, in the genomicregion of the gene of interest or 10 kb up- or downstream of the codingregion of a gene in a configuration such that the T-DNA inhibitsexpression of the targeted gene. Typically, regulation of expression ofthe targeted gene by its natural promoter is disrupted. The T-DNA israndomly inserted into the plant genome, for example, throughAgrobacterium infection and leads to down-regulated expression of genesnear the inserted T-DNA. The resulting transgenic plants show phenotypesdue to inhibited expression of genes close to the introduced T-DNA.

A genetic modification may also be introduced in the locus of a MADS15gene using the technique of TILLING (Targeted Induced Local Lesions InGenomes).

Site-directed mutagenesis and random mutagenesis may be used to generatevariants of MADS15 nucleic acids. Several methods are available toachieve site-directed mutagenesis, the most common being PCR basedmethods (current protocols in molecular biology. Wiley Eds.).

Directed evolution may also be used to generate variants of MADS15nucleic acids. This consists of iterations of DNA shuffling followed byappropriate screening and/or selection to generate variants of MADS15nucleic acids or variants thereof encoding MADS15 polypeptides having amodified (here decreased or abolished) biological activity (Castle etal., (2004) Science 304(5674): 1151-4; U.S. Pat. Nos. 5,811,238 and6,395,547).

T-DNA activation, TILLING, site-directed mutagenesis and directedevolution are examples of technologies that enable the generation ofnovel alleles and MADS15 variants.

The effects of the invention may also be reproduced using homologousrecombination. The nucleic acid to be targeted may be an allele encodingan inactive protein or a protein with decreased activity, used toreplace the endogenous gene or may be introduced in addition to theendogenous gene, and needs to be targeted to the locus of the MADS15gene.

Other methods, such as the use of antibodies directed to the endogenousMADS15 for inhibiting its function in planta, or interference in thesignalling pathway in which MADS15 is involved, will be well known tothe skilled man. Alternatively, a screening program may be set up toidentify natural variants of a MADS15 gene, which variants have reducedMADS15 activity, or no MADS15 activity at all. Such natural variants mayalso be used in the methods of the present invention.

For optimal performance, the gene silencing techniques used for thereduction or substantial elimination of endogenous MADS15 geneexpression requires the use of MADS15 nucleic acid sequences frommonocotyledonous plants for transformation into monocotyledonous plants.Preferably, a MADS15 nucleic acid from any given plant species isintroduced into that same species. For example, a MADS15 nucleic acidfrom rice (be it a full length MADS15 sequence or a fragment) istransformed into a rice plant. The MADS15 nucleic acid need not beintroduced into the same plant variety.

Reference herein to a “MADS15 gene” or a “MADS15 nucleic acid” is takento mean a polymeric form of a deoxyribonucleotide or a ribonucleotidepolymer of any length, either double- or single-stranded, or analoguesthereof, that have the essential characteristic of a naturalribonucleotide in that they can hybridise to nucleic acids in a mannersimilar to naturally occurring polynucleotides. A “MADS15 gene” or a“MADS15 nucleic acid” refers to a sufficient length of substantiallycontiguous nucleotides of a MADS15-encoding gene to perform genesilencing; this may be as little as 20 or fewer nucleotides. A geneencoding a (functional) protein is not a requirement for the variousmethods discussed above for the reduction or substantial elimination ofexpression of an endogenous MADS15 gene.

The methods of the invention may be performed using a sufficient lengthof substantially contiguous nucleotides of a MADS15 gene/nucleic acid,which may consist of 20 or fewer nucleotides, which may be from any partof the MADS15 gene/nucleic acid, such as the 5′ end of the coding regionthat is well conserved amongst the MADS15 gene family, or encoding oneof the conserved motifs described below.

MADS15 genes are well known in the art and useful in the methods of theinvention are substantially contiguous nucleotides of the plant MADS15genes/nucleic acid described in Moon et al. (Plant Physiol. 120,1193-1204, 1999).

Other MADS15 gene/nucleic acid sequences may also be used in the methodsof the invention, and may readily be identified by a person skilled inthe art. MADS15 polypeptides may be identified by the presence of one ormore of several well-known features (see below). Upon identification ofa MADS15 polypeptide, a person skilled in the art could easily derive,using routine techniques, the corresponding encoding nucleic acidsequence and use a sufficient length of contiguous nucleotides of thesame to perform any one or more of the gene silencing methods describedabove.

The term “MADS15 polypeptide or homologue thereof” as defined hereinrefers to a protein that falls in the group of FUL-like MADS boxproteins as delineated by Adam et al. (J. Mol. Evol. 62, 15-31).FUL-like MADS box proteins are part of the SQUAMOSA subfamily and havethe typical MIKC^(c) architecture: a MADS domain (M) at the N-terminus,followed by an intervening domain (I) involved in dimerisation, a highlyconserved keratin domain (K) and a variable domain (C) at theC-terminus, which is responsible for binding of interacting proteins ortranscriptional activation (De Bodt et al. Trends in Plant Science 8,475-483).

Plant MADS15 polypeptides may also be identified by the presence ofcertain conserved motifs. The presence of these conserved motifs may beidentified using methods for the alignment of sequences for comparisonas described hereinabove. In some instances, the default parameters maybe adjusted to modify the stringency of the search. For example usingBLAST, the statistical significance threshold (called “expect” value)for reporting matches against database sequences may be increased toshow less stringent matches. This way, short nearly exact matches may beidentified. Upon identification of a MADS15 polypeptide by the presenceof these motifs, a person skilled in the art may easily derive thecorresponding nucleic acid encoding the polypeptide comprising therelevant motifs, and use a sufficient length of contiguous nucleotidesof the same to perform any one or more of the gene silencing methodsdescribed above (for the reduction or substantial elimination of anendogenous MADS15 gene expression).

Typically, the presence of at least one of the motifs 1 to 4 should besufficient to identify any query sequence as a MADS15, however thepresence of at least motifs 1 and 2 is preferred. The consensus sequenceprovided is based on the monocot sequences given in the sequencelisting. A person skilled in the art would be well aware that theconsensus sequence may vary somewhat if further or different sequences(for example from dicot sequences) were used for comparison.

Motif 1, located in the MADS domain (SEQ ID NO: 114):L (L/V) KKA (H/N) EIS (VI) L (C/Y) DAE (V/I/L) (A/G) (L/A/V) (I/V) (I/V) FS(T/P/A/N) KGKLYE (Y/F) (A/S) (T/S) (D/N/E) S (K/C/R/S) M (D/E) (N/I/R/K) IL(E/D) R. Preferably, this conserved sequence motif 1 has the sequence:LLKKAHEISVLCDAEVA (L/A/V) I (I/V) FS (T/P) KGKLYEYATDS (C/R) M (D/E) (R/K)ILER, most preferably the conserved sequence motif 1 has the sequence:LLKKAHEISVLCDAEVAAIVFSPKGKLYEYATDSRMDKILERMotif 2, located in the K domain (SEQ ID NO: 115):KLK (A/S) (K/R) (V/I) E (A/T/S) (L/I) (Q/N) (K/R/N) (S/C/R) (Q/H) (R/K)HLMGE Preferably, this conserved sequence motif 2 has the sequence:KLKAK (V/I) E (A/T) (L/I) QK (S/C) (Q/H) (R/K) HLMGEMore preferably, this conserved sequence motif 2 has the sequence:KLKAKIETIQKCHKHLMGE

Optionally, the MADS15 polypeptide or its homologue may comprise in theC domain motif 3 and/or motif 4:

motif 3 (SEQ ID NO: 116): Q (P/Q/V/A) QTS (S/F) (S/F) (S/F) (S/F)(S/C/F) (F/M) motif 4 (SEQ ID NO: 117): (G/A/V/L) (L/P) XWMX (S/H)wherein the first X residue in motif 4 may be any amino acid, butpreferably L, P or H; and wherein the second X residue may be any aminoacid, but preferably V or L.

Alternatively, the C-domain (starting behind the keratin domain, i.e. atR175 in SEQ ID NO: 110) may be characterised by the increased occurrenceof glutamine (normally 3.93%, here at least 8.77% with a maximum of26.73%), in addition, the content in alanine, proline, and/or serine mayalso be increased (above 7.8%, 4.85% and 6.89% respectively).

Alternatively formulated, a preferred MADS15 protein useful in themethods of the present invention comprises at least an N-terminal MADSdomain corresponding to the SMART domain SM00432 (Pfam PF00319) followedby a Keratin domain corresponding to the K-box region defined in Pfam asPF01486, more preferably, the MADS15 protein has in its MADS domain theconserved signature of SEQ ID NO: 114 and in its K-domain the conservedsignature of SEQ ID NO: 115, most preferably, the MADS15 protein has thesequence given in SEQ ID NO: 110.

Homologues, as defined above, may readily be identified using routinetechniques well known in the art, such as by sequence alignment;homologues of OsMADS15 may have been named differently in various plantspecies, therefore the gene/protein names should not be used foridentifying orthologues or paralogues. Methods for the alignment ofsequences for comparison are well known in the art, such methods includeGAP, BESTFIT, BLAST, FASTA and TFASTA. GAP uses the algorithm ofNeedleman and Wunsch ((1970) J Mol Biol 48: 443-453) to find thealignment of two complete sequences that maximizes the number of matchesand minimizes the number of gaps. The BLAST algorithm (Altschul et al.(1990) J Mol Biol 215: 403-10) calculates percent sequence identity andperforms a statistical analysis of the similarity between the twosequences. The software for performing BLAST analysis is publiclyavailable through the National Centre for Biotechnology Information.Homologous sequences may readily be identified using, for example, theClustalW multiple sequence alignment algorithm (version 1.83), with thedefault pairwise alignment parameters, and a scoring method inpercentage. Minor manual editing may be performed to optimise alignmentbetween conserved motifs (see below), as would be apparent to a personskilled in the art.

The various structural domains in a MADS15 protein may be identifiedusing specialised databases e.g. SMART (Schultz et al. (1998) Proc.Natl. Acad. Sci. USA 95, 5857-5864; Letunic et al. (2002) Nucleic AcidsRes 30, 242-244), InterPro (Mulder et al., (2003) Nucl. Acids. Res. 31,315-318;), Prosite (Bucher and Bairoch (1994), A generalized profilesyntax for biomolecular sequences motifs and its function in automaticsequence interpretation. (In) ISMB-94; Proceedings 2nd InternationalConference on Intelligent Systems for Molecular Biology. Altman R.,Brutlag D., Karp P., Lathrop R., Searls D., Eds., pp 53-61, AAAIPress,Menlo Park; Hulo et al., Nucl. Acids. Res. 32:D134-D137, (2004)) or Pfam(Bateman et al., Nucleic Acids Research 30(1): 276-280 (2002)).

Furthermore, a MADS15 protein may also be identifiable by its ability tobind DNA and to interact with other proteins. DNA-binding activity andprotein-protein interactions may readily be determined in vitro or invivo using techniques well known in the art. Examples of in vitro assaysfor DNA binding activity include: gel retardation analysis using knownMADS-box DNA binding domains (West et al. (1998) Nucl Acid Res 26(23):5277-87), or yeast one-hybrid assays. An example of an in vitro assayfor protein-protein interactions is the yeast two-hybrid analysis(Fields and Song (1989) Nature 340:245-6). Proteins known to interactwith OsMADS15 include other MADS proteins (such as those involved in theflowering signalling complex), receptor like kinases such a Clavata1 andClavata2, Erecta, BRI1 or RSK.

Therefore upon identification of a MADS15 polypeptide using one orseveral of the features described above, a person skilled in the art mayeasily derive the corresponding nucleic acid encoding the polypeptide,and use a sufficient length of substantially contiguous nucleotides ofthe same to perform any one or more of the gene silencing methodsdescribed above (for the reduction or substantial elimination of anendogenous MADS15 gene expression).

Preferred for use in the methods of the invention is a sufficient lengthof substantially contiguous nucleotides of SEQ ID NO: 109 (OsMADS15), orthe use of a sufficient length of substantially contiguous nucleotidesof a nucleic acid sequence encoding an orthologue or paralogue ofOsMADS15 (SEQ ID NO: 109). Examples of such orthologues and paraloguesof OsMADS15 are provided in Table D below. Close homologues of OsMADS15are the proteins represented by SEQ ID NO: 147, 149, 151, 153, 155, 157,159, 161, 163, 165, 167, 169, 171 and 173.

Orthologues in, for example, monocot plant species may easily be foundby performing a so-called reciprocal blast search. This may be done by afirst blast involving blasting a query sequence (for example, SEQ ID NO:109 or SEQ ID NO: 110) against any sequence database, such as thepublicly available NCBI database. BLASTN or TBLASTX (using standarddefault values) may be used when starting from a nucleotide sequence andBLASTP or TBLASTN (using standard default values) may be used whenstarting from a protein sequence. The BLAST results may optionally befiltered. The full-length sequences of either the filtered results ornon-filtered results are then BLASTed back (second BLAST) againstsequences from the organism from which the query sequence is derived(where the query sequence is SEQ ID NO: 109 or SEQ ID NO: 110 the secondblast would therefore be against rice sequences). The results of thefirst and second BLASTs are then compared. A paralogue is identified ifa high-ranking hit from the first blast is from the same species as fromwhich the query sequence is derived; an orthologue is identified if ahigh-ranking hit is not from the same species as from which the querysequence is derived. High-ranking hits are those having a low E-value.The lower the E-value, the more significant the score (or in other wordsthe lower the chance that the hit was found by chance). Computation ofthe E-value is well known in the art. In the case of large families,ClustalW may be used, followed by a neighbour joining tree, to helpvisualize clustering of related genes and to identify orthologues andparalogues.

The source of the substantially contiguous nucleotides of a MADS15gene/nucleic acid may be any plant source or artificial source. Foroptimal performance, the gene silencing techniques used for thereduction or substantial elimination of endogenous MADS15 geneexpression requires the use of MADS15 sequences from monocotyledonousplants for transformation into monocotyledonous plants. Preferably,MADS15 sequences from the family Poaceae are transformed into plants ofthe family Poaceae. Further preferably, a MADS15 nucleic acid from rice(be it a full length MADS15 sequence or a fragment) is transformed intoa rice plant. The MADS15 nucleic acid need not be introduced into thesame plant variety. Most preferably, the MADS15 nucleic acid from riceis a sufficient length of substantially contiguous nucleotides of SEQ IDNO: 109 (OsMADS15) or a sufficient length of substantially contiguousnucleotides of a nucleic acid sequence encoding an orthologue orparalogue of OsMADS15 (SEQ ID NO: 110). As mentioned above, a personskilled in the art would be well aware of what would constitute asufficient length of substantially contiguous nucleotides to perform anyof the gene silencing methods defined hereinabove, this may be as littleas 20 or fewer substantially contiguous nucleotides in some cases.

The invention also provides genetic constructs and vectors to facilitateintroduction and/or expression of the nucleotide sequences useful in themethods according to the invention.

Therefore, there is provided a gene construct comprising one or morecontrol sequences capable of driving expression of a sense and/orantisense MADS15 nucleic acid sequence in a plant so as to silence anendogenous MADS15 gene in the plant; and optionally a transcriptiontermination sequence. Preferably, the control sequence is a constitutiveand ubiquitous promoter.

A preferred construct for gene silencing is one comprising an invertedrepeat of a MADS15 gene or fragment thereof, preferably capable offorming a hairpin structure, which inverted repeat is under the controlof a constitutive promoter.

Therefore, the invention provides a construct comprising:

-   -   (a) a MADS15 nucleic acid capable of forming a hairpin        structure;    -   (b) one or more control sequences capable of driving expression        of the nucleic acid sequence of (a); and optionally    -   (c) a transcription termination sequence.

Constructs useful in the methods according to the present invention maybe created using recombinant DNA technology well known to personsskilled in the art. The gene constructs may be inserted into vectors,which may be commercially available, suitable for transforming intoplants and suitable for expression of the gene of interest in thetransformed cells. The invention therefore provides use of a geneconstruct as defined hereinabove in the methods of the invention.

The sequence of interest is operably linked to one or more controlsequences (at least to a promoter) capable of increasing expression in aplant.

Advantageously, any type of promoter may be used to drive expression ofthe nucleic acid sequence. The promoter may be an inducible promoter,i.e. having induced or increased transcription initiation in response toa developmental, chemical, environmental or physical stimulus.Additionally or alternatively, the promoter may be a tissue-preferred orcell-preferred promoter, i.e. one that is capable of preferentiallyinitiating transcription in certain tissues, such as the leaves, roots,seed tissue etc, or even in specific cells. Promoters able to initiatetranscription in certain tissues or cells only are referred to herein as“tissue-specific”, respectively “cell-specific”.

Preferably, the MADS15 nucleic acid or functional variant thereof isoperably linked to a constitutive promoter. Preferably the promoter is aubiquitous promoter and is expressed predominantly throughout the plant.Preferably, the constitutive promoter capable of preferentiallyexpressing the nucleic acid throughout the plant has a comparableexpression profile to a GOS2 promoter. More preferably, the constitutivepromoter has the same expression profile as the rice GOS2 promoter, mostpreferably, the promoter capable of preferentially expressing thenucleic acid throughout the plant is the GOS2 promoter from rice (SEQ IDNO: 113 or SEQ ID NO: 174). It should be clear that the applicability ofthe present invention is not restricted to the MADS15 nucleic acidrepresented by SEQ ID NO: 109, nor is the applicability of the inventionrestricted to expression of a MADS15 nucleic acid when driven by a GOS2promoter. An alternative constitutive promoter that is useful in themethods of the present invention is the high mobility group proteinpromoter (PRO0170, SEQ ID NO: 40 in WO2004070039). Examples of otherconstitutive promoters that may also be used to drive expression of aMADS15 nucleic acid are shown in the definitions section.

Optionally, one or more terminator sequences may also be used in theconstruct introduced into a plant. Additional regulatory elements mayinclude transcriptional as well as translational enhancers. Thoseskilled in the art will be aware of terminator and enhancer sequencesthat may be suitable for use in performing the invention. Such sequenceswould be known or may readily be obtained by a person skilled in theart.

The genetic constructs of the invention may further include an origin ofreplication sequence that is required for maintenance and/or replicationin a specific cell type. One example is when a genetic construct isrequired to be maintained in a bacterial cell as an episomal geneticelement (e.g. plasmid or cosmid molecule). Preferred origins ofreplication include, but are not limited to, the f1-ori and colE1. Thegenetic construct may optionally comprise a selectable marker gene.

The present invention also encompasses plants including plant partsobtainable by the methods according to the present invention havingincreased yield relative to control plants and which have reduced orsubstantially eliminated expression of an endogenous MADS15 gene.

The invention furthermore provides a method for the production oftransgenic plants having increased yield relative to control plants,which transgenic plants have reduced or substantially eliminatedexpression of an endogenous MADS15 gene.

More specifically, the present invention provides a method for theproduction of transgenic plants having increased seed yield which methodcomprises:

-   -   introducing and expressing in a plant, plant part or plant cell        a gene construct comprising one or more control sequences        capable of preferentially driving expression of an inverted        repeat MADS15 nucleic acid sequence in a plant so as to silence        an endogenous MADS15 gene in the plant; and    -   cultivating the plant, plant part or plant cell under conditions        promoting plant growth and development.

Preferably, the construct introduced into a plant is one comprising aninverted repeat (in part or complete) of a MADS15 gene or fragmentthereof, preferably capable of forming a hairpin structure.

According to a preferred feature of the present invention, the constructis introduced into a plant by transformation.

Generally after transformation, plant cells or cell groupings areselected for the presence of one or more markers which are encoded byplant-expressible genes co-transferred with the gene of interest,following which the transformed material is regenerated into a wholeplant.

Following DNA transfer and regeneration, putatively transformed plantsmay be evaluated, for instance using Southern analysis, for the presenceof the gene of interest, copy number and/or genomic organisation.Alternatively or additionally, expression levels of the newly introducedDNA may be monitored using Northern and/or Western analysis, orquantitative PCR, all techniques being well known to persons havingordinary skill in the art.

The generated transformed plants may be propagated by a variety ofmeans, such as by clonal propagation or classical breeding techniques.For example, a first generation (or T1) transformed plant may be selfedto give homozygous second generation (or T2) transformants, and the T2plants further propagated through classical breeding techniques.

The generated transformed organisms may take a variety of forms. Forexample, they may be chimeras of transformed cells and non-transformedcells; clonal transformants (e.g., all cells transformed to contain theexpression cassette); grafts of transformed and untransformed tissues(e.g., in plants, a transformed rootstock grafted to an untransformedscion).

The present invention clearly extends to any plant cell or plantproduced by any of the methods described herein, and to all plant partsand propagules thereof. The present invention extends further toencompass the progeny of a primary transformed or transfected cell,tissue, organ or whole plant that has been produced by any of theaforementioned methods, the only requirement being that progeny exhibitthe same genotypic and/or phenotypic characteristic(s) as those producedby the parent in the methods according to the invention.

The invention also extends to harvestable parts of a plant such as seedsand products derived, preferably directly derived, from a harvestablepart of such a plant, such as dry pellets or powders, oil, fat and fattyacids, starch or proteins.

The present invention also encompasses use of MADS15 nucleic acids forthe reduction or substantial elimination of endogenous MADS15 geneexpression in a plant for increasing plant seed yield as definedhereinabove.

Nucleic acids encoding the MADS15 protein described herein, or theMADS15 proteins themselves, may find use in breeding programmes in whicha DNA marker is identified which may be genetically linked to aMADS15-encoding gene. The nucleic acids/genes, or the MADS15 proteinsthemselves may be used to define a molecular marker. This DNA or proteinmarker may then be used in breeding programmes to select plants havingenhanced yield-related traits as defined hereinabove in the methods ofthe invention.

Allelic variants of a MADS15 protein-encoding nucleic acid/gene may alsofind use in marker-assisted breeding programmes. Such breedingprogrammes sometimes require introduction of allelic variation bymutagenic treatment of the plants, using for example EMS mutagenesis;alternatively, the programme may start with a collection of allelicvariants of so called “natural” origin caused unintentionally.Identification of allelic variants then takes place, for example, byPCR. This is followed by a step for selection of superior allelicvariants of the sequence in question and which give increased yield.Selection is typically carried out by monitoring growth performance ofplants containing different allelic variants of the sequence inquestion. Growth performance may be monitored in a greenhouse or in thefield. Further optional steps include crossing plants in which thesuperior allelic variant was identified with another plant. This couldbe used, for example, to make a combination of interesting phenotypicfeatures.

Nucleic acids encoding MADS15 proteins may also be used as probes forgenetically and physically mapping the genes that they are a part of,and as markers for traits linked to those genes. Such information may beuseful in plant breeding in order to develop lines with desiredphenotypes. Such use of MADS15 protein-encoding nucleic acids requiresonly a nucleic acid sequence of at least 15 nucleotides in length. TheMADS15 protein-encoding nucleic acids may be used as restrictionfragment length polymorphism (RFLP) markers. Southern blots (Sambrook J,Fritsch E F and Maniatis T (1989) Molecular Cloning, A LaboratoryManual) of restriction-digested plant genomic DNA may be probed with theMADS15 protein-encoding nucleic acids. The resulting banding patternsmay then be subjected to genetic analyses using computer programs suchas MapMaker (Lander et al. (1987) Genomics 1: 174-181) in order toconstruct a genetic map. In addition, the nucleic acids may be used toprobe Southern blots containing restriction endonuclease-treated genomicDNAs of a set of individuals representing parent and progeny of adefined genetic cross. Segregation of the DNA polymorphisms is noted andused to calculate the position of the MADS15 protein-encoding nucleicacid in the genetic map previously obtained using this population(Botstein et al. (1980) Am. J. Hum. Genet. 32:314-331).

The production and use of plant gene-derived probes for use in geneticmapping is described in Bernatzky and Tanksley (1986) Plant Mol. Biol.Reporter 4: 37-41. Numerous publications describe genetic mapping ofspecific cDNA clones using the methodology outlined above or variationsthereof. For example, F2 intercross populations, backcross populations,randomly mated populations, near isogenic lines, and other sets ofindividuals may be used for mapping. Such methodologies are well knownto those skilled in the art.

The nucleic acid probes may also be used for physical mapping (i.e.,placement of sequences on physical maps; see Hoheisel et al. In:Non-mammalian Genomic Analysis: A Practical Guide, Academic press 1996,pp. 319-346, and references cited therein).

In another embodiment, the nucleic acid probes may be used in directfluorescence in situ hybridisation (FISH) mapping (Trask (1991) TrendsGenet. 7:149-154). Although current methods of FISH mapping favour useof large clones (several kb to several hundred kb; see Laan et al.(1995) Genome Res. 5:13-20), improvements in sensitivity may allowperformance of FISH mapping using shorter probes.

A variety of nucleic acid amplification-based methods for genetic andphysical mapping may be carried out using the nucleic acids. Examplesinclude allele-specific amplification (Kazazian (1989) J. Lab. Clin. Med11:95-96), polymorphism of PCR-amplified fragments (CAPS; Sheffield etal. (1993) Genomics 16:325-332), allele-specific ligation (Landegren etal. (1988) Science 241:1077-1080), nucleotide extension reactions(Sokolov (1990) Nucleic Acid Res. 18:3671), Radiation Hybrid Mapping(Walter et al. (1997) Nat. Genet. 7:22-28) and Happy Mapping (Dear andCook (1989) Nucleic Acid Res. 17:6795-6807). For these methods, thesequence of a nucleic acid is used to design and produce primer pairsfor use in the amplification reaction or in primer extension reactions.The design of such primers is well known to those skilled in the art. Inmethods employing PCR-based genetic mapping, it may be necessary toidentify DNA sequence differences between the parents of the mappingcross in the region corresponding to the instant nucleic acid sequence.This, however, is generally not necessary for mapping methods.

The methods according to the present invention result in plants havingenhanced yield-related traits, as described hereinbefore. These traitsmay also be combined with other economically advantageous traits, suchas further yield-enhancing traits, tolerance to other abiotic and bioticstresses, traits modifying various architectural features and/orbiochemical and/or physiological features.

Detailed Description of PLT

Surprisingly, it has now been found that increasing expression of anucleic acid encoding a PLT transcription factor polypeptide givesplants having enhanced yield-related traits, in particular increasedyield relative to control plants.

According to the present invention, there is provided a method forincreasing plant yield relative to control plants, comprising increasingexpression in a plant of a nucleic acid encoding a PLT transcriptionfactor polypeptide.

The term “increased yield” as defined herein is taken to mean anincrease in biomass (weight) of one or more parts of a plant, which mayinclude aboveground (harvestable) parts and/or (harvestable) parts belowground.

In particular, such harvestable parts are seeds, and performance of themethods of the invention results in plants having increased seed yieldrelative to the seed yield of control plants.

Taking corn as an example, a yield increase may be manifested as one ormore of the following: increase in the number of plants per hectare oracre, an increase in the number of ears per plant, an increase in thenumber of rows, number of kernels per row, kernel weight, thousandkernel weight, ear length/diameter, increase in the seed filling rate(which is the number of filled seeds divided by the total number ofseeds and multiplied by 100), among others. Taking rice as an example, ayield increase may manifest itself as an increase in one or more of thefollowing: number of plants per hectare or acre, number of panicles perplant, number of spikelets per panicle, number of flowers (florets) perpanicle (which is expressed as a ratio of the number of filled seedsover the number of primary panicles), increase in the seed filling rate(which is the number of filled seeds divided by the total number ofseeds and multiplied by 100), increase in thousand kernel weight, amongothers.

According to a preferred feature of the present invention, performanceof the methods of the invention gives plants having increased seed yieldrelative to control plants. Therefore according to the presentinvention, there is provided a method for increasing seed yield inplants relative to the seed yield of control plants, the methodcomprising increasing expression in a plant of a nucleic acid encoding aPLT transcription factor polypeptide.

Since the transgenic plants according to the present invention haveincreased yield, it is likely that these plants exhibit an increasedgrowth rate (during at least part of their life cycle), relative to thegrowth rate of corresponding wild type plants at a corresponding stagein their life cycle. The increased growth rate may be specific to one ormore parts of a plant (including seeds), or may be throughoutsubstantially the whole plant. A plant having an increased growth ratemay even exhibit early flowering. The increase in growth rate may takeplace at one or more stages in the life cycle of a plant or duringsubstantially the whole plant life cycle. Increased growth rate duringthe early stages in the life cycle of a plant may reflect enhancedvigour (increased seedling vigor at emergence). The increase in growthrate may alter the harvest cycle of a plant allowing plants to be sownlater and/or harvested sooner than would otherwise be possible. If thegrowth rate is sufficiently increased, it may allow for the furthersowing of seeds of the same plant species (for example sowing andharvesting of rice plants followed by sowing and harvesting of furtherrice plants all within one conventional growing period). Similarly, ifthe growth rate is sufficiently increased, it may allow for the furthersowing of seeds of different plants species (for example the sowing andharvesting of corn plants followed by, for example, the sowing andoptional harvesting of soy bean, potato or any other suitable plant).Harvesting additional times from the same rootstock in the case of somecrop plants may also be possible. Altering the harvest cycle of a plantmay lead to an increase in annual biomass production per acre (due to anincrease in the number of times (say in a year) that any particularplant may be grown and harvested). An increase in growth rate may alsoallow for the cultivation of transgenic plants in a wider geographicalarea than their wild-type counterparts, since the territoriallimitations for growing a crop are often determined by adverseenvironmental conditions either at the time of planting (early season)or at the time of harvesting (late season). Such adverse conditions maybe avoided if the harvest cycle is shortened. The growth rate may bedetermined by deriving various parameters from growth curves, suchparameters may be: T-Mid (the time taken for plants to reach 50% oftheir maximal size) and T-90 (time taken for plants to reach 90% oftheir maximal size), amongst others.

Performance of the methods of the invention gives plants having anincreased growth rate. Therefore, according to the present invention,there is provided a method for increasing the growth rate of plants,which method comprises increasing expression in a plant of a nucleicacid encoding a PLT transcription factor polypeptide.

An increase in yield and/or growth rate occurs whether the plant isunder non-stress conditions or whether the plant is exposed to variousstresses compared to control plants. Plants typically respond toexposure to stress by growing more slowly. In conditions of severestress, the plant may even stop growing altogether. Mild stress on theother hand is defined herein as being any stress to which a plant isexposed which does not result in the plant ceasing to grow altogetherwithout the capacity to resume growth. Mild stress in the sense of theinvention leads to a reduction in the growth of the stressed plants ofless than 40%, 35% or 30%, preferably less than 25%, 20% or 15%, morepreferably less than 14%, 13%, 12%, 11% or 10% or less in comparison tothe control plant under non-stress conditions. Due to advances inagricultural practices (irrigation, fertilization, pesticide treatments)severe stresses are not often encountered in cultivated crop plants. Asa consequence, the compromised growth induced by mild stress is often anundesirable feature for agriculture. Mild stresses are the everydaybiotic and/or abiotic (environmental) stresses to which a plant isexposed. Abiotic stresses may be due to drought or excess water,anaerobic stress, salt stress, chemical toxicity, oxidative stress andhot, cold or freezing temperatures. The abiotic stress may be an osmoticstress caused by a water stress (particularly due to drought), saltstress, oxidative stress or an ionic stress. Biotic stresses aretypically those stresses caused by pathogens, such as bacteria, viruses,fungi and insects.

In particular, the methods of the present invention may be performedunder non-stress conditions or under conditions of mild drought to giveplants having increased yield relative to control plants. As reported inWang et al. (Planta (2003) 218: 1-14), abiotic stress leads to a seriesof morphological, physiological, biochemical and molecular changes thatadversely affect plant growth and productivity. Drought, salinity,extreme temperatures and oxidative stress are known to be interconnectedand may induce growth and cellular damage through similar mechanisms.Rabbani et al. (Plant Physiol (2003) 133: 1755-1767) describes aparticularly high degree of “cross talk” between drought stress andhigh-salinity stress. For example, drought and/or salinisation aremanifested primarily as osmotic stress, resulting in the disruption ofhomeostasis and ion distribution in the cell. Oxidative stress, whichfrequently accompanies high or low temperature, salinity or droughtstress, may cause denaturing of functional and structural proteins. As aconsequence, these diverse environmental stresses often activate similarcell signaling pathways and cellular responses, such as the productionof stress proteins, up-regulation of anti-oxidants, accumulation ofcompatible solutes and growth arrest. The term “non-stress” conditionsas used herein are those environmental conditions that allow optimalgrowth of plants. Persons skilled in the art are aware of normal soilconditions and climatic conditions for a given location.

Performance of the methods of the invention gives plants grown undernon-stress conditions or under mild drought conditions enhancedyield-related traits relative to control plants grown under comparableconditions. Therefore, according to the present invention, there isprovided a method for enhancing yield-related traits, in particular forincreasing yield, in plants grown under non-stress conditions or undermild drought conditions, which method comprises increasing expression ina plant of a nucleic acid encoding a PLT transcription factorpolypeptide.

The methods of the invention are advantageously applicable to any plant.

Plants that are particularly useful in the methods of the inventioninclude all plants which belong to the superfamily Viridiplantae, inparticular monocotyledonous and dicotyledonous plants including fodderor forage legumes, ornamental plants, food crops, trees or shrubs.According to a preferred embodiment of the present invention, the plantis a crop plant. Examples of crop plants include soybean, sunflower,canola, alfalfa, rapeseed, cotton, tomato, potato and tobacco. Examplesof typical crop plants grown for oil production include soybean,sunflower, cotton, canola, peanuts or palm. Examples of typical cropplants grown for starch production include rice, wheat, barley, corn orpotato. Further preferably, the plant is a monocotyledonous plant.Examples of monocotyledonous plants include sugarcane. More preferablythe plant is a cereal. Examples of cereals include rice, maize, wheat,barley, millet, rye, triticale, sorghum and oats.

The term “PLT transcription factor polypeptide” as defined herein refersto any polypeptide comprising in increasing order of preference at least70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity tothe AP2 domain represented by SEQ ID NO: 191.

Preferably the PLT transcription factor polypeptide of choice is a PLTtranscription factor polypeptide that in its natural genetic environmentis essentially expressed in the roots (below ground parts) of the plant.

Further preferably, the PLT transcription factor polypeptide compriseseither one motif but preferably both of motif1 as represented by SEQ IDNO: 192 PK(V/L)(A/E)DFLG and motif 2 as represented by SEQ ID NO: 209:(V/L)FX(M/V)WN(D/E), wherein X may be any amino acid; preferably motif 2has the sequence of SEQ ID NO: 193 (V/L)F(T/S/N)(M/V)WN(D/E).

Examples of PLT transcription factor polypeptides comprising (i) inincreasing order of preference at least 70%, 75%, 80%, 85%, 90%, 95%,96%, 97%, 98% or 99% sequence identity to the AP2 domain represented bySEQ ID NO: 191 are proteins represented by the sequences given in TableI in the examples section. Preferred examples are represented by SEQ IDNO: 176 and SEQ ID NO: 178.

The AP2 domain in a PLT transcription factor polypeptide may beidentified using specialised databases e.g. SMART (Schultz et al. (1998)Proc. Natl. Acad. Sci. USA 95, 5857-5864; Letunic et al. (2002) NucleicAcids Res 30, 242-244), InterPro (Mulder et al., (2003) Nucl. Acids.Res. 31, 315-318), Prosite (Bucher and Bairoch (1994), A generalizedprofile syntax for biomolecular sequences motifs and its function inautomatic sequence interpretation. (In) ISMB-94; Proceedings 2ndInternational Conference on Intelligent Systems for Molecular Biology.Altman R., Brutlag D., Karp P., Lathrop R., Searls D., Eds., pp 53-61,AAAIPress, Menlo Park; Hulo et al., Nucl. Acids. Res. 32:D134-D137,(2004)) or Pfam (Bateman et al., Nucleic Acids Research 30(1): 276-280(2002)). The AP2 domain of a PLT transcription factor comprises tworepeats R1 and R2, each of about 68 amino acids and separated by alinker region (FIG. 13).

The AP2 domain as represented by SEQ ID NO: 191 may be identified usingmethods for the alignment of sequences for comparison as describedhereinabove. In some instances, the default parameters may be adjustedto modify the stringency of the search. For example using BLAST, thestatistical significance threshold (called “expect” value) for reportingmatches against database sequences may be increased to show lessstringent matches. This way, short nearly exact matches may beidentified, including motif1 as represented by SEQ ID NO: 192 and motif2 as represented by SEQ ID NO: 209, preferably as represented by SEQ IDNO: 193.

Homologues of a PLT transcription factor polypeptide may also be used toperform the methods of invention. Homologues (or homologous proteins)may readily be identified using routine techniques well known in theart, such as by sequence alignment. Methods for the alignment ofsequences for comparison are well known in the art, such methods includeGAP, BESTFIT, BLAST, FASTA and TFASTA. GAP uses the algorithm ofNeedleman and Wunsch ((1970) J Mol Biol 48: 443-453) to find thealignment of two complete sequences that maximizes the number of matchesand minimizes the number of gaps. The BLAST algorithm (Altschul et al.(1990) J Mol Biol 215: 403-10) calculates percent sequence identity andperforms a statistical analysis of the similarity between the twosequences. The software for performing BLAST analysis is publiclyavailable through the National Centre for Biotechnology Information.Homologues may readily be identified using, for example, the ClustalWmultiple sequence alignment algorithm (version 1.83), with the defaultpairwise alignment parameters, and a scoring method in percentage. Minormanual editing may be performed to optimise alignment between conservedmotifs, as would be apparent to a person skilled in the art.

Homologues also include orthologues and paralogues. Orthologues andparalogues may easily be found by performing a so-called reciprocalblast search. This may be done by a first BLAST involving BLASTing aquery sequence (for example, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO:177 or SEQ ID NO: 178) against any sequence database, such as thepublicly available NCBI database. BLASTN or TBLASTX (using standarddefault values) may be used when starting from a nucleotide sequence andBLASTP or TBLASTN (using standard default values) may be used whenstarting from a protein sequence. The BLAST results may optionally befiltered. The full-length sequences of either the filtered results ornon-filtered results are then BLASTed back (second BLAST) againstsequences from the organism from which the query sequence is derived(where the query sequence is SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO:177 or SEQ ID NO: 178, the second BLAST would therefore be againstArabidopsis thaliana sequences). The results of the first and secondBLASTs are then compared. A paralogue is identified if a high-rankinghit from the first BLAST is from the same species as from which thequery sequence is derived; an orthologue is identified if a high-rankinghit is not from the same species as from which the query sequence isderived. High-ranking hits are those having a low E-value. The lower theE-value, the more significant the score (or in other words the lower thechance that the hit was found by chance). Computation of the E-value iswell known in the art. In the case of large families, ClustalW may beused, followed by a neighbour joining tree, to help visualize clusteringof related genes and to identify orthologues and paralogues. In additionto E-values, comparisons are also scored by percentage identity.Percentage identity refers to the number of identical nucleotides (oramino acids) between the two compared nucleic acid (or polypeptide)sequences over a particular length. PLT transcription factor polypeptidehomologues have in increasing order of preference at least 25%, 30%,40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or moresequence identity or similarity (functional identity) to an unmodifiedPLT transcription factor polypeptide as represented by SEQ ID NO: 176 orSEQ ID NO: 178. Percentage identity between PLT transcription factorpolypeptide homologues outside of the AP2 domain is reputedly low.Preferably, PLT transcription factor polypeptide homologues comprise anAP2 domain with in increasing order of preference at least 70%, 75%,80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the AP2domain represented by SEQ ID NO: 191. Further preferably, PLTtranscription factor polypeptide homologues are as represented by SEQ IDNO: 180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO: 186, SEQ ID NO: 188,SEQ ID NO: 200 and SEQ ID NO: 202.

The PLT polypeptide may be a derivative of SEQ ID NO: 176 or SEQ ID NO:178. “Derivatives” include peptides, oligopeptides, polypeptides whichmay, compared to the amino acid sequence of the naturally-occurring formof the protein, such as the one presented in SEQ ID NO: 176 or SEQ IDNO: 178, comprise substitutions of amino acids with non-naturallyoccurring amino acid residues, or additions of non-naturally occurringamino acid residues. Derivatives of SEQ ID NO: 180, SEQ ID NO: 182, SEQID NO: 184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO: 200 and SEQ IDNO: 202 are further examples which may be suitable for use in themethods of the invention provided that they have the same or similarbiological activity.

Furthermore, PLT transcription factor polypeptides (at least in theirnative form) typically have DNA-binding activity and an activationdomain. A person skilled in the art may easily determine the presence ofan activation domain and DNA-binding activity using routine techniquesand procedures. Proteins interacting with PLT transcription factorpolypeptides (as, for example, in transcriptional complexes) may easilybe identified using standard techniques for a person skilled in the art.

Examples of nucleic acids encoding PLT transcription factor polypeptidesinclude but are not limited to those represented by any one of: SEQ IDNO: 175, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO: 181, SEQ ID NO: 183,SEQ ID NO: 185, SEQ ID NO: 187, SEQ ID NO: 199 and SEQ ID NO: 201.Variants of nucleic acids encoding PLT transcription factor polypeptidesmay be suitable for use in the methods of the invention provided thatthey have the same or similar biological activity. Suitable variantsinclude portions of nucleic acids encoding PLT transcription factorpolypeptides and/or nucleic acids capable of hybridising with nucleicacids/genes encoding PLT transcription factor polypeptides. Furthervariants include splice variants and allelic variants of nucleic acidsencoding PLT transcription factor polypeptides.

The term “portion” as defined herein refers to a piece of DNA encoding apolypeptide comprising in increasing order of preference at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to theAP2 domain represented by SEQ ID NO: 191. The portion may furthercomprise either one motif but preferably both of motif1 as representedby SEQ ID NO: 192 and/or motif 2 as represented by SEQ ID NO: 209;preferably, motif 2 has the sequences as represented by SEQ ID NO: 193.

A portion may be prepared, for example, by making one or more deletionsto a nucleic acid encoding a PLT transcription factor polypeptide. Theportions may be used in isolated form or they may be fused to othercoding (or non coding) sequences in order to, for example, produce aprotein that combines several activities. When fused to other codingsequences, the resulting polypeptide produced upon translation may bebigger than that predicted for the PLT transcription factor portion.Preferably, the portion codes for a polypeptide with substantially thesame biological activity as the PLT transcription factor polypeptides ofSEQ ID NO: 176 and SEQ ID NO: 178.

Preferably, the portion is a portion of a nucleic acid as represented byany one of the sequences listed in Table I of the Examples. Mostpreferably the portion is a portion of a nucleic acid as represented bySEQ ID NO: 175 and SEQ ID NO: 177.

Another variant of a nucleic acid encoding a PLT transcription factorpolypeptide, useful in the methods of the present invention, is anucleic acid capable of hybridising under reduced stringency conditions,preferably under stringent conditions, with a probe derived from thenucleic acid as defined hereinbefore, which hybridising sequence encodesa polypeptide comprising in increasing order of preference at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to theAP2 domain represented by SEQ ID NO: 191. The hybridizing sequence mayfurther comprise either one motif but preferably both of motif1 asrepresented by SEQ ID NO: 192 and/or motif 2 as represented by SEQ IDNO: 209, preferably as represented by SEQ ID NO: 193.

Preferably, the hybridising sequence is one that is capable ofhybridising to a nucleic acid as represented by (or to probes derivedfrom) any one of the sequences listed in Table I of the Examples, or toa portion of any of the aforementioned sequences (the target sequence).Most preferably the hybridising sequence is capable of hybridising toSEQ ID NO: 175 or to SEQ ID NO: 177 (or to probes derived therefrom).Probes are generally less than 1000 bp in length, preferably less than500 bp in length. Commonly, probe lengths for DNA-DNA hybridisationssuch as Southern blotting, vary between 100 and 500 bp, whereas thehybridising region in probes for DNA-DNA hybridisations such as in PCRamplification generally are shorter than 50 but longer than 10nucleotides.

The PLT transcription factor polypeptide may be encoded by a splicevariant. The term “splice variant” as used herein encompasses variantsof a nucleic acid sequence in which selected introns and/or exons havebeen excised, replaced, displaced or added, or in which introns havebeen shortened or lengthened. Such variants will be ones in which thesubstantial biological activity of the protein is retained, which may beachieved by selectively retaining functional segments of the protein.Such splice variants may be found in nature or may be manmade. Methodsfor making such splice variants are well known in the art.

Preferred splice variants are splice variants of the nucleic acidencoding a PLT transcription factor polypeptide comprising in increasingorder of preference at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%or 99% sequence identity to the AP2 domain represented by SEQ ID NO:191. Splice variants may further comprise either one motif butpreferably both of motif1 as represented by SEQ ID NO: 192 and/or motif2 as represented by SEQ ID NO: 209, preferably as represented by SEQ IDNO: 193.

Further preferred splice variants of nucleic acids encoding PLTtranscription factor polypeptides comprising features as definedhereinabove are splice variants of a nucleic acid as represented by anyone of the sequences listed in Table I of the Examples. Most preferredis a splice variant of a nucleic acid sequence as represented by SEQ IDNO: 175 and SEQ ID NO: 177.

The PLT transcription factor polypeptide may also be encoded by anallelic variant of a nucleic acid encoding a polypeptide comprising fromcomprising in increasing order of preference at least 70%, 75%, 80%,85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the AP2 domainrepresented by SEQ ID NO: 191. The allelic variant may further compriseeither one motif but preferably both of motif1 as represented by SEQ IDNO: 192 and/or motif 2 as represented by SEQ ID NO: 209, preferably asrepresented by SEQ ID NO: 193.

Preferred allelic variants of nucleic acids encoding PLT transcriptionfactor polypeptides comprising features as defined hereinabove aresplice variants of a nucleic acid as represented by any one of thesequences listed in Table I of the Examples. Most preferred is anallelic variant of a nucleic acid sequence as represented by SEQ ID NO:175 and SEQ ID NO: 177.

The increase in expression of a nucleic acid encoding a PLTtranscription factor polypeptide leads to raised corresponding mRNA orpolypeptide levels, which could equate to raised activity of the PLTtranscription factor polypeptide; or the activity may also be raisedwhen there is no change in polypeptide levels, or even when there is areduction in polypeptide levels. This may occur when the intrinsicproperties of the polypeptide are altered, for example, by making mutantversions that are more active that the wild type polypeptide.

Directed evolution (or gene shuffling) may be used to generate variantsof nucleic acids encoding PLT transcription factor polypeptides. Thisconsists of iterations of DNA shuffling followed by appropriatescreening and/or selection to generate variants of nucleic acids orportions thereof encoding PLT transcription factor polypeptides orhomologues or portions thereof having an modified biological activity(Castle et al., (2004) Science 304(5674): 1151-4; U.S. Pat. Nos.5,811,238 and 6,395,547).

Site-directed mutagenesis may be used to generate variants of nucleicacids encoding PLT transcription factor polypeptides. Several methodsare available to achieve site-directed mutagenesis, the most commonbeing PCR based methods (current protocols in molecular biology. WileyEds.).

Directed evolution and site-directed mutagenesis are examples oftechnologies that enable the generation of variants of nucleic acidsencoding PLT transcription factor polypeptides with modified activityuseful to perform the methods of the invention.

Nucleic acids encoding PLT transcription factor polypeptides may bederived from any natural or artificial source. The nucleic acid may bemodified from its native form in composition and/or genomic environmentthrough deliberate human manipulation. Preferably the PLT transcriptionfactor nucleic acid is from a plant, preferably from a dicotyledonousplant, further preferably from the Brassicaceae family, more preferablyfrom the Arabidopsis genus, most preferably the nucleic acid is fromArabidopsis thaliana.

The methods of the invention rely on increased expression of a nucleicacid encoding a PLT transcription factor polypeptide in a plant. Thenucleic acid may be a full-length nucleic acid or may be a portion or ahybridising sequence or another nucleic acid variant as hereinbeforedefined.

The methods of the invention rely on increased expression of a nucleicacid encoding a PLT transcription factor polypeptide in a plant.

The invention also provides genetic constructs and vectors to facilitateintroduction and/or expression in a plant of the nucleic acid sequencesuseful in the methods according to the invention.

Therefore, there is provided a gene construct comprising:

-   -   (i) A nucleic acid encoding a PLT transcription factor        polypeptide as defined hereinabove;    -   (ii) One or more control sequences, of which at least one is a        medium strength constitutive promoter.

Constructs useful in the methods according to the present invention maybe constructed using recombinant DNA technology well known to personsskilled in the art. The gene constructs may be inserted into vectors,which may be commercially available, suitable for transforming intoplants and suitable for expression of the gene of interest in thetransformed cells. The invention therefore provides use of a geneconstruct as defined hereinabove in the methods of the invention.

Plants are transformed with a vector comprising the sequence of interest(i.e., a nucleic acid encoding a PLT transcription factor polypeptide).The skilled artisan is well aware of the genetic elements that must bepresent on the vector in order to successfully transform, select andpropagate host cells containing the sequence of interest. The sequenceof interest is operably linked to one or more control sequences (atleast to a promoter).

The nucleic acid encoding a PLT transcription factor polypeptide orvariant thereof is operably linked to a constitutive promoter,preferably a medium strength constitutive promoter. A constitutivepromoter is transcriptionally active during most, but not necessarilyall, phases of its growth and development and is substantiallyubiquitously expressed at moderate levels. Promoter strength and/orexpression pattern can be analysed as described in the definitionssection. The promoter strength and/or expression pattern can for examplebe compared to that of a well-characterised shoot preferred referencepromoter, such as the Cab27 promoter (weak expression, GenBank AP004700)or the putative protochlorophyllid reductase promoter (strongexpression, GenBank AL606456). Preferably the promoter used in themethods of the present invention is derived from a plant, furtherpreferably a monocotyledonous plant. Most preferred is use of a GOS2promoter (from rice) (SEQ ID NO: 194 or alternatively SEQ ID NO: 210).It should be clear that the applicability of the present invention isnot restricted to the nucleic acid represented by SEQ ID NO: 175 or SEQID NO: 177, nor is the applicability of the invention restricted toexpression of a nucleic acid encoding a PLT transcription factorpolypeptide when driven by a GOS2 promoter. Examples of otherconstitutive promoters that may also be used to drive expression of anucleic acid encoding a PLT transcription factor polypeptide are shownin the definitions section.

Optionally, one or more terminator sequences (also a control sequence)may be used in the construct introduced into a plant. Additionalregulatory elements may include transcriptional as well as translationalenhancers. Those skilled in the art will be aware of terminator andenhancer sequences that may be suitable for use in performing theinvention. Other control sequences (besides promoter, enhancer,silencer, intron sequences, 3′UTR and/or 5′UTR regions) may be proteinand/or RNA stabilizing elements. Such sequences would be known or mayreadily be obtained by a person skilled in the art.

The genetic constructs of the invention may further include an origin ofreplication sequence that is required for maintenance and/or replicationin a specific cell type. One example is when a genetic construct isrequired to be maintained in a bacterial cell as an episomal geneticelement (e.g. plasmid or cosmid molecule). Preferred origins ofreplication include, but are not limited to, the f1-ori and colE1. Thegenetic construct may optionally comprise a selectable marker gene.

The present invention also encompasses plants obtainable by the methodsaccording to the present invention. The present invention thereforeprovides plants, plant parts or plant cells thereof obtainable by themethod according to the present invention, which plants or parts orcells thereof comprise a nucleic acid transgene encoding a PLTtranscription factor polypeptide under the control of a constitutivepromoter, preferably a non-viral constitutive promoter.

The invention also provides a method for the production of transgenicplants having increased yield relative to control plants, comprisingintroduction and preferential expression of a nucleic acid encoding aPLT transcription factor polypeptide in a plant.

More specifically, the present invention provides a method for theproduction of transgenic plants having increased yield which methodcomprises:

-   -   (i) introducing and expressing a nucleic acid encoding a PLT        transcription factor polypeptide in a plant; and    -   (ii) cultivating the plant cell under conditions promoting plant        growth and development.

The nucleic acid may be introduced directly into a plant cell or intothe plant itself (including introduction into a tissue, organ or anyother part of a plant). According to a preferred feature of the presentinvention, the nucleic acid is preferably introduced into a plant bytransformation.

Generally after transformation, plant cells or cell groupings areselected for the presence of one or more markers which are encoded byplant-expressible genes co-transferred with the gene of interest,following which the transformed material is regenerated into a wholeplant. Following DNA transfer and regeneration, putatively transformedplants may be evaluated, for instance using Southern analysis, for thepresence of the gene of interest, copy number and/or genomicorganisation. Alternatively or additionally, expression levels of thenewly introduced DNA may be monitored using Northern and/or Westernanalysis, or quantitative PCR, all techniques being well known topersons having ordinary skill in the art.

The generated transformed plants may be propagated by a variety ofmeans, such as by clonal propagation or classical breeding techniques.For example, a first generation (or T1) transformed plant may be selfedto give homozygous second generation (or T2) transformants, and the T2plants further propagated through classical breeding techniques.

The generated transformed organisms may take a variety of forms. Forexample, they may be chimeras of transformed cells and non-transformedcells; clonal transformants (e.g., all cells transformed to contain theexpression cassette); grafts of transformed and untransformed tissues(e.g., in plants, a transformed rootstock grafted to an untransformedscion).

The present invention clearly extends to any plant cell or plantproduced by any of the methods described herein, and to all plant partsand propagules thereof. The present invention extends further toencompass the progeny of a primary transformed or transfected cell,tissue, organ or whole plant that has been produced by any of theaforementioned methods, the only requirement being that progeny exhibitthe same genotypic and/or phenotypic characteristic(s) as those producedby the parent in the methods according to the invention.

The invention also includes host cells containing an isolated nucleicacid encoding a PLT transcription factor polypeptide. Preferred hostcells according to the invention are plant cells.

The invention furthermore extends to harvestable parts of a plant suchas, but not limited to seeds, leaves, fruits, flowers, stems, rhizomes,tubers and bulbs. The invention furthermore relates to products derived,preferably directly derived, from a harvestable part of such a plant,such as dry pellets or powders, oil, fat and fatty acids, starch orproteins.

The present invention also encompasses use of nucleic acids encoding PLTtranscription factor polypeptides and use of PLT transcription factorpolypeptides in increasing plant yield as defined hereinabove in themethods of the invention.

The increased expression of a nucleic acid encoding a PLT transcriptionfactor polypeptide may be performed by introducing a geneticmodification (preferably in the locus of a PLT transcription factorgene). The locus of a gene as defined herein is taken to mean a genomicregion, which includes the gene of interest and 10 KB up- or downstreamof the coding region.

The genetic modification may be introduced by alternative methods as theone described hereinabove, for example, by any one (or more) of thefollowing: T-DNA activation, TILLING and homologous recombination.Following introduction of the genetic modification, there follows anoptional step of selecting for increased expression of a nucleic acidencoding a PLT transcription factor polypeptide, which increasedexpression gives plants having increased yield.

T-DNA activation tagging results in transgenic plants that show dominantphenotypes due to modified expression of genes close to the introducedpromoter. The promoter to be introduced a medium strength constitutivepromoter capable of increasing expression of the nucleic acid encoding aPLT transcription factor polypeptide in a plant.

A genetic modification may also be introduced in the locus of a geneencoding a PLT transcription factor polypeptide using the technique ofTILLING (Targeted Induced Local Lesions In Genomes).

The effects of the invention may also be reproduced using homologousrecombination. The nucleic acid to be introduced (which may be a nucleicacid encoding a PLT transcription factor polypeptide or variant thereofas hereinbefore defined) is targeted to the locus of a PLT gene. Thenucleic acid to be targeted may be an improved allele used to replacethe endogenous gene or may be introduced in addition to the endogenousgene.

T-DNA activation, TILLING and homologous recombination are examples oftechnologies that enable the generation of genetic modificationscomprising increasing expression of a nucleic acid encoding a PLTtranscription factor polypeptide in plants.

Nucleic acids encoding PLT transcription factor polypeptides, or PLTtranscription factor polypeptides, may find use in breeding programmesin which a DNA marker is identified which may be genetically linked to aPLT transcription factor gene. The nucleic acids/genes, or the PLTtranscription factor polypeptides may be used to define a molecularmarker. This DNA or protein marker may then be used in breedingprogrammes to select plants having increased yield as definedhereinabove in the methods of the invention.

Allelic variants of a PLT transcription factor nucleic acid/gene mayalso find use in marker-assisted breeding programmes. Such breedingprogrammes sometimes require introduction of allelic variation bymutagenic treatment of the plants, using for example EMS mutagenesis;alternatively, the programme may start with a collection of allelicvariants of so called “natural” origin caused unintentionally.Identification of allelic variants then takes place, for example, byPCR. This is followed by a step for selection of superior allelicvariants of the sequence in question and which give increased yield.Selection is typically carried out by monitoring growth performance ofplants containing different allelic variants of the sequence inquestion. Growth performance may be monitored in a greenhouse or in thefield. Further optional steps include crossing plants in which thesuperior allelic variant was identified with another plant. This couldbe used, for example, to make a combination of interesting phenotypicfeatures.

A nucleic acid encoding a PLT transcription factor polypeptide may alsobe used as probes for genetically and physically mapping the genes thatthey are a part of, and as markers for traits linked to those genes.Such information may be useful in plant breeding in order to developlines with desired phenotypes. Such use of PLT transcription factornucleic acids requires only a nucleic acid sequence of at least 15nucleotides in length. The PLT transcription factor nucleic acids may beused as restriction fragment length polymorphism (RFLP) markers.Southern blots (Sambrook J, Fritsch E F and Maniatis T (1989) MolecularCloning, A Laboratory Manual) of restriction-digested plant genomic DNAmay be probed with the PLT transcription factor nucleic acids. Theresulting banding patterns may then be subjected to genetic analysesusing computer programs such as MapMaker (Lander et al. (1987) Genomics1: 174-181) in order to construct a genetic map. In addition, thenucleic acids may be used to probe Southern blots containing restrictionendonuclease-treated genomic DNAs of a set of individuals representingparent and progeny of a defined genetic cross. Segregation of the DNApolymorphisms is noted and used to calculate the position of the PLTtranscription factor nucleic acid in the genetic map previously obtainedusing this population (Botstein et al. (1980) Am. J. Hum. Genet.32:314-331).

The production and use of plant gene-derived probes for use in geneticmapping is described in Bernatzky and Tanksley (1986) Plant Mol. Biol.Reporter 4: 37-41. Numerous publications describe genetic mapping ofspecific cDNA clones using the methodology outlined above or variationsthereof. For example, F2 intercross populations, backcross populations,randomly mated populations, near isogenic lines, and other sets ofindividuals may be used for mapping. Such methodologies are well knownto those skilled in the art.

The nucleic acid probes may also be used for physical mapping (i.e.,placement of sequences on physical maps; see Hoheisel et al. In:Non-mammalian Genomic Analysis: A Practical Guide, Academic press 1996,pp. 319-346, and references cited therein).

In another embodiment, the nucleic acid probes may be used in directfluorescence in situ hybridisation (FISH) mapping (Trask (1991) TrendsGenet. 7:149-154). Although current methods of FISH mapping favor use oflarge clones (several kb to several hundred kb; see Laan et al. (1995)Genome Res. 5:13-20), improvements in sensitivity may allow performanceof FISH mapping using shorter probes.

A variety of nucleic acid amplification-based methods for genetic andphysical mapping may be carried out using the nucleic acids. Examplesinclude allele-specific amplification (Kazazian (1989) J. Lab. Clin. Med11:95-96), polymorphism of PCR-amplified fragments (CAPS; Sheffield etal. (1993) Genomics 16:325-332), allele-specific ligation (Landegren etal. (1988) Science 241:1077-1080), nucleotide extension reactions(Sokolov (1990) Nucleic Acid Res. 18:3671), Radiation Hybrid Mapping(Walter et al. (1997) Nat. Genet. 7:22-28) and Happy Mapping (Dear andCook (1989) Nucleic Acid Res. 17:6795-6807). For these methods, thesequence of a nucleic acid is used to design and produce primer pairsfor use in the amplification reaction or in primer extension reactions.The design of such primers is well known to those skilled in the art. Inmethods employing PCR-based genetic mapping, it may be necessary toidentify DNA sequence differences between the parents of the mappingcross in the region corresponding to the instant nucleic acid sequence.This, however, is generally not necessary for mapping methods.

The methods according to the present invention result in plants havingincreased yield, as described hereinbefore. This increased yield mayalso be combined with other economically advantageous traits, such asfurther yield-enhancing traits, tolerance to other abiotic and bioticstresses, traits modifying various architectural features and/orbiochemical and/or physiological features.

Detailed Description of bHLH

Surprisingly, it has now been found that modulating expression in aplant of a nucleic acid encoding a particular class of bHLHtranscription factor gives plants having enhanced yield-related traitsrelative to control plants. The particular class of bHLH transcriptionfactor suitable for enhancing yield-related traits in plants isdescribed in detail below.

The present invention provides a method for enhancing yield-relatedtraits in plants relative to control plants, comprising modulatingexpression in a plant of a nucleic acid encoding a particular class ofbHLH transcription factor.

A preferred method for modulating (preferably, increasing) expression ofa nucleic acid encoding a bHLH transcription factor is by introducingand expressing in a plant a nucleic acid encoding a particular class ofbHLH transcription factor as further defined below.

The nucleic acid to be introduced into a plant (and therefore useful inperforming the methods of the invention) is any nucleic acid encodingthe type of bHLH transcription factor which will now be described. AbHLH transcription factor as defined herein refers to a polypeptiderepresented by any one of SEQ ID NO 213, SEQ ID NO 215, SEQ ID NO: 217,SEQ ID NO: 219, SEQ ID NO 225, SEQ ID NO: 297, SEQ ID NO: 299 and SEQ IDNO: 301. The invention is illustrated by transforming plants with theOryza sativa sequence represented by SEQ ID NO: 212, encoding thepolypeptide of SEQ ID NO: 213. SEQ ID NO: 215 from Oryza sativa (encodedby SEQ ID NO: 214) and SEQ ID NO: 217 from Oryza sativa (encoded by SEQID NO: 216) are paralogues of the polypeptide of SEQ ID NO: 213. SEQ IDNO: 219 from Arabidopsis thaliana (encoded by SEQ ID NO: 218) and SEQ IDNO: 225 from Arabidopsis thaliana (encoded by SEQ ID NO: 224) areorthologues of the polypeptide of SEQ ID NO: 213. SEQ ID NO: 221 fromArabidopsis thaliana (encoded by SEQ ID NO: 220) and SEQ ID NO: 223 fromArabidopsis thaliana (encoded by SEQ ID NO: 222) are variants of SEQ IDNO: 218 and SEQ ID NO: 219.

Orthologues and paralogues may easily be found by performing a so-calledreciprocal blast search. Typically this involves a first BLAST involvingBLASTing a query sequence (for example, SEQ ID NO: 212, SEQ ID NO: 213,SEQ ID NO: 214, SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID NO: 217, SEQ IDNO: 218, SEQ ID NO: 219, SEQ ID NO: 224 or SEQ ID NO: 225) against anysequence database, such as the publicly available NCBI database. BLASTNor TBLASTX (using standard default values) are generally used whenstarting from a nucleotide sequence, and BLASTP or TBLASTN (usingstandard default values) when starting from a protein sequence. TheBLAST results may optionally be filtered. The full-length sequences ofeither the filtered results or non-filtered results are then BLASTedback (second BLAST) against sequences from the organism from which thequery sequence is derived (where the query sequence is SEQ ID NO: 212,SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, SEQ ID NO: 216 or SEQ IDNO: 217, the second BLAST would therefore be against Oryza sequences;where the query sequence is SEQ ID NO: 218, SEQ ID NO: 219, SEQ ID NO:224 or SEQ ID NO: 225, the second BLAST would therefore be againstArabidopsis sequences). The results of the first and second BLASTs arethen compared. A paralogue is identified if a high-ranking hit from thefirst blast is from the same species as from which the query sequence isderived, a BLAST back then ideally results in the query sequence ashighest hit; an orthologue is identified if a high-ranking hit in thefirst BLAST is not from the same species as from which the querysequence is derived, and preferably results upon BLAST back in the querysequence being among the highest hits.

High-ranking hits are those having a low E-value. The lower the E-value,the more significant the score (or in other words the lower the chancethat the hit was found by chance). Computation of the E-value is wellknown in the art. In addition to E-values, comparisons are also scoredby percentage identity. Percentage identity refers to the number ofidentical nucleotides (or amino acids) between the two compared nucleicacid (or polypeptide) sequences over a particular length. Preferably thescore is greater than 50, more preferably greater than 100; andpreferably the E-value is less than e-5, more preferably less than e-6.An example detailing the identification of orthologues and paralogues isgiven in Example 31 and Example 30 respectively herein. In the case oflarge families, ClustalW may be used, followed by a neighbour joiningtree, to help visualize clustering of related genes and to identifyorthologues and paralogues.

Examples of orthologues obtained by the BLAST procedure mentioned aboveare SEQ ID NO: 227 from Medicago truncatula (encoded by SEQ ID NO: 226)and SEQ ID NO: 229 from Hordeum vulgare (encoded by SEQ ID NO: 228).Further orthologues and paralogues may readily be identified using theBLAST procedure described above and by following the procedure given inthe Examples section.

The proteins represented by SEQ ID NO: 297, 299 and 301 were hithertounknown. Therefore, the invention also provides hitherto unknown bHLHtranscription factors and bHLH transcription factor-encoding nucleicacids.

According to a further embodiment of the present invention, there istherefore provided an isolated nucleic acid molecule comprising:

-   -   (i) a nucleic acid represented by any one of SEQ ID NO: 296, SEQ        ID NO: 298 and SEQ ID NO: 300;    -   (ii) the complement of a nucleic acid represented by any one of        SEQ ID NO: 296, SEQ ID NO: 298 and SEQ ID NO: 300;    -   (iii) a nucleic acid encoding a bHLH transcription factor        having (a) in increasing order of preference, at least 50%, 55%,        60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or        more sequence identity to the amino acid sequence represented by        any one of SEQ ID NO: 297, SEQ ID NO: 299 and SEQ ID NO: 301,        and (b) a bHLH domain.

According to a further embodiment of the present invention, there isalso provided an isolated polypeptide comprising:

-   -   (i) an amino acid sequence represented by any one of SEQ ID NO:        297, SEQ ID NO: 299 and SEQ ID NO: 301;    -   (ii) an amino acid sequence having (a) in increasing order of        preference, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,        90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any        one of the amino acid sequences represented by SEQ ID NO: 297,        SEQ ID NO: 299 and SEQ ID NO: 301, and (b) a bHLH domain;    -   (iii) derivatives of any of the amino acid sequences given        in (i) or (ii) above.

The polypeptides represented by any one of SEQ ID NO 213, SEQ ID NO 215,SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO 221, SEQ ID NO 223, SEQ ID NO225, SEQ ID NO: 227, SEQ ID NO: 229, SEQ ID NO: 297, SEQ ID NO: 299, SEQID NO: 301, or orthologues or paralogues of any of the aforementionedSEQ ID NOs, all comprise a bHLH domain.

bHLH domains are well known in the art and may readily be identified bypersons skilled in the art. The family is defined by a bHLH signaturedomain, which consists of 60 or so amino acids with two functionallydistinct regions. A basic region, located at the N-terminal end of thedomain, is involved in DNA binding and consists of 15 or so amino acidswith a high number of basic residues. An HLH region, at the C-terminalend, functions as a dimerization domain and mainly comprises hydrophobicresidues that form two amphipathic helices separated by a loop region ofvariable sequence and length.

A bHLH domain may be identified using methods for the alignment ofsequences for comparison. In some instances, default parameters may beadjusted to modify the stringency of the search. For example usingBLAST, the statistical significance threshold (called E-value) forreporting matches against database sequences may be increased to showless stringent matches. In this way, short nearly exact matches may beidentified.

Methods for the alignment of sequences for comparison are well known inthe art, such methods include GAP, BESTFIT, BLAST, FASTA and TFASTA. GAPuses the algorithm of Needleman and Wunsch ((1970) J Mol Biol 48:443-453) to find the global (over the whole the sequence) alignment oftwo sequences that maximizes the number of matches and minimizes thenumber of gaps. The BLAST algorithm (Altschul et al. (1990) J Mol Biol215: 403-10) calculates percent sequence identity and performs astatistical analysis of the similarity between the two sequences. Thesoftware for performing BLAST analysis is publicly available through theNational Centre for Biotechnology Information (NCBI). Homologues mayreadily be identified using, for example, the ClustalW multiple sequencealignment algorithm (version 1.83), with the default pairwise alignmentparameters, and a scoring method in percentage. Minor manual editing maybe performed to optimise alignment between conserved motifs, as would beapparent to a person skilled in the art.

Specialist databases also exist for the identification of domains. ThebHLH domain in a bHLH transcription factor may be identified using, forexample, SMART (Schultz et al. (1998) Proc. Natl. Acad. Sci. USA 95,5857-5864; Letunic et al. (2002) Nucleic Acids Res 30, 242-244),InterPro (Mulder et al., (2003) Nucl. Acids. Res. 31, 315-318), Prosite(Bucher and Bairoch (1994), A generalized profile syntax forbiomolecular sequences motifs and its function in automatic sequenceinterpretation. (In) ISMB-94; Proceedings 2nd International Conferenceon Intelligent Systems for Molecular Biology. Altman R., Brutlag D.,Karp P., Lathrop R., Searls D., Eds., pp 53-61, AAAIPress, Menlo Park;Hulo et al., Nucl. Acids. Res. 32:D134-D137, (2004)) or Pfam (Bateman etal., Nucleic Acids Research 30(1): 276-280 (2002)). The HLH domainstructure in the InterPro database is designated IPR001092 andIPR011598; PF00010 in the Pfam database; SM00353 in the SMART databaseand PS50888 in the PROSITE database. Furthermore, the alignment shown inFIG. 18 highlights the bHLH domain of bHLH transcription factors usefulin the methods of the invention.

The polypeptides represented by any one of SEQ ID NO 213, SEQ ID NO 215,SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO 225, SEQ ID NO: 297, SEQ IDNO: 299, SEQ ID NO: 301 or orthologues or paralogues of any of theaforementioned SEQ ID NOs, typically exhibit considerable sequencedivergence outside of the conserved bHLH domain.

FIG. 19 a is a matrix showing the overall similarities and identities(in bold) of the bHLH type proteins described above. Even though theidentities appear to be relatively low, polypeptides having sequenceidentity falling within the ranges shown in the matrix may be taken tobe orthologues or paralogues of any of SEQ ID NO 213, SEQ ID NO 215, SEQID NO: 217, SEQ ID NO: 219 or SEQ ID NO 225; this may be confirmed bythe reciprocal blast procedure described above. Typically, nucleic acidsencoding bHLH transcription factors useful in the methods of theinvention have, in increasing order of preference, at least 13%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95% or more sequence identity to the bHLH transcription factorsrepresented by any one of SEQ ID NO 213, SEQ ID NO 215, SEQ ID NO: 217,SEQ ID NO: 219 or SEQ ID NO 225.

The matrix shown in FIG. 19 b shows similarities and identities (inbold) over the bHLH domain, where of course the values are higher thanwhen considering full length proteins. Typically, nucleic acids encodingbHLH transcription factors useful in the methods of the invention havebHLH domains having, in increasing order of preference, at least 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% ormore sequence identity to the bHLH domain of any one of the polypeptidesrepresented by any one of SEQ ID NO 213, SEQ ID NO 215, SEQ ID NO: 217,SEQ ID NO: 219 or SEQ ID NO 225.

A pattern of amino acids, termed a 5-9-13 configuration, may be found atthree positions within the basic region of the bHLH domain (see FIG. 4of Heim et al., 2003 (Mol. Biol. Evol. 20(5):735-747) and FIG. 18 hereinwhere three upwardly pointing arrows show the configuration). Thepolypeptides represented by any one of SEQ ID NO 213, SEQ ID NO 215, SEQID NO: 217, SEQ ID NO: 219, SEQ ID NO 225, SEQ ID NO: 297, SEQ ID NO:299, SEQ ID NO: 301 or orthologues or paralogues of any of theaforementioned SEQ ID NOs, preferably comprise a K/R ER configuration,more preferably an RER configuration, within the bHLH domain, typicallywithin the basic region of the domain. The presence of thisconfiguration is not a prerequisite to performing the methods of theinvention, therefore some variation in the K/R ER configuration isacceptable. Arabidopsis bHLH polypeptides were grouped into twelvesubfamilies according to structural similarities (see FIG. 4 of Heim etal., 2003). Members of group IX constitute bHLH polypeptides having anRER configuration. Furthermore, one member of Group VI comprises an RERconfiguration.

The polypeptides represented by any one of SEQ ID NO 213, SEQ ID NO 215,SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO 225, SEQ ID NO: 297, SEQ IDNO: 299, SEQ ID NO: 301 or orthologues or paralogues of any of theaforementioned SEQ ID NOs, may also comprise (in addition to a bHLHdomain and optionally a K/R ER 5-9-13 motif, preferably an RER motif) adomain designated PFB26111 (see Pfam database). The domain is alsoindicated in FIG. 18.

Typically, nucleic acids encoding bHLH transcription factors (as definedabove) having a bHLH domain and having, in increasing order ofpreference, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% ormore sequence identity to the PFB26111 domain (see FIG. 18) of any oneof the polypeptides represented by any one of SEQ ID NO 213, SEQ ID NO215, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO 225, SEQ ID NO: 297, SEQID NO: 299, or SEQ ID NO: 301 are useful in the methods of theinvention.

Nucleic acids encoding the polypeptides represented by any one of SEQ IDNO 213, SEQ ID NO 215, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO 225,SEQ ID NO: 297, SEQ ID NO: 299, SEQ ID NO: 301, or orthologues orparalogues of any of the aforementioned SEQ ID NOs, need not befull-length nucleic acids, since performance of the methods of theinvention does not rely on the use of full length nucleic acidsequences. Examples of nucleic acids suitable for use in performing themethods of the invention include but are not limited to thoserepresented by any one of: SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO:216, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO: 224, SEQID NO: 226, SEQ ID NO: 228, SEQ ID NO: 296, SEQ ID NO: 298, and SEQ IDNO: 300. Nucleic acid variants may also be useful in practising themethods of the invention. Examples of such nucleic acid variants includeportions of nucleic acids encoding a bHLH transcription factor asdefined herein, splice variants of nucleic acids encoding a bHLHtranscription factor as defined herein, allelic variants of nucleicacids encoding a bHLH transcription factor as defined herein andvariants of nucleic acids encoding a bHLH transcription factor asdefined herein that are obtained by gene shuffling. The terms portion,splice variant, allelic variant and gene shuffling will now bedescribed.

A portion of a nucleic acids encoding a bHLH transcription factor asdefined herein may be prepared, for example, by making one or moredeletions to the nucleic acid. The portions may be used in isolated formor they may be fused to other coding (or non coding) sequences in orderto, for example, produce a protein that combines several activities.When fused to other coding sequences, the resultant polypeptide producedupon translation may be bigger than that predicted for the bHLHtranscription factor portion.

According to the present invention, there is provided a method forenhancing yield-related traits in plants, comprising introducing andexpressing in a plant a portion of a nucleic acid encoding a bHLHtranscription factor represented by any of SEQ ID NO 213, SEQ ID NO 215,SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO 225, SEQ ID NO: 297, SEQ IDNO: 299, SEQ ID NO: 301, or a portion of a nucleic acid encodingorthologues, paralogues or homologues of any of the aforementioned SEQID NOs.

Portions useful in the methods of the invention, encode a polypeptidehaving a bHLH domain (as described above) and having substantially thesame biological activity as the bHLH transcription factor represented byany of SEQ ID NO 213, SEQ ID NO 215, SEQ ID NO: 217, SEQ ID NO: 219, SEQID NO 225, SEQ ID NO: 297, SEQ ID NO: 299, SEQ ID NO: 301, ororthologues or paralogues of any of the aforementioned SEQ ID NOs. Theportion is typically at least 150 consecutive nucleotides in length,preferably at least 300 consecutive nucleotides in length, morepreferably at least 400 consecutive nucleotides in length and mostpreferably at least 500 consecutive nucleotides in length. Preferably,the portion is a portion of a nucleic acid as represented by any one ofSEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO: 216, SEQ ID NO: 218, SEQ IDNO: 220, SEQ ID NO: 222, SEQ ID NO: 224, SEQ ID NO: 226, SEQ ID NO: 228,SEQ ID NO: 296, SEQ ID NO: 298, and SEQ ID NO: 300. Most preferably theportion is a portion of a nucleic acid as represented by SEQ ID NO: 212.

Another nucleic acid variant useful in the methods of the invention, isa nucleic acid capable of hybridising under reduced stringencyconditions, preferably under stringent conditions, with a nucleic acidencoding a bHLH transcription factor as defined herein, or a with aportion as defined herein.

Hybridising sequences useful in the methods of the invention, encode apolypeptide having a bHLH domain (as described above) and havingsubstantially the same biological activity as the bHLH transcriptionfactor represented by any of SEQ ID NO 213, SEQ ID NO 215, SEQ ID NO:217, SEQ ID NO: 219, SEQ ID NO 225, SEQ ID NO: 297, SEQ ID NO: 299, SEQID NO: 301 or having substantially the same biological activity asorthologues or paralogues of any of the aforementioned SEQ ID NOs. Thehybridising sequence is typically at least 150 consecutive nucleotidesin length, preferably at least 300 consecutive nucleotides in length,more preferably at least 400 consecutive nucleotides in length and mostpreferably at least 500 consecutive nucleotides in length. Preferably,the hybridising sequence is one that is capable of hybridising to any ofthe nucleic acids represented by SEQ ID NO: 212, SEQ ID NO: 214, SEQ IDNO: 216, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO: 224,SEQ ID NO: 226, SEQ ID NO: 228, SEQ ID NO: 296, SEQ ID NO: 298, and SEQID NO: 300 or to a portion of any of the aforementioned sequences, aportion being as defined above. Most preferably the hybridising sequenceis capable of hybridising to a nucleic acid as represented by SEQ ID NO:212, or to portions thereof.

According to the present invention, there is provided a method forenhancing yield-related traits in plants, comprising introducing andexpressing in a plant a nucleic acid capable of hybridizing to a nucleicacid encoding a bHLH transcription factor represented by any of SEQ IDNO 213, SEQ ID NO 215, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO 225,SEQ ID NO: 297, SEQ ID NO: 299, SEQ ID NO: 301, or comprisingintroducing and expressing in a plant a nucleic acid capable ofhybridising to a nucleic acid encoding an orthologue, paralogue orhomologue of any of the aforementioned SEQ ID NOs.

Another nucleic acid variant useful in the methods of the invention is asplice variant encoding a bHLH transcription factor as definedhereinabove.

According to the present invention, there is provided a method forenhancing yield-related traits in plants, comprising introducing andexpressing in a plant a splice variant of a nucleic acid encoding a bHLHtranscription factor represented by any of SEQ ID NO 213, SEQ ID NO 215,SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO 225, SEQ ID NO: 297, SEQ IDNO: 299, and SEQ ID NO: 301, or a splice variant of a nucleic acidencoding an orthologue, paralogue or homologue of any of theaforementioned SEQ ID NOs.

Preferred splice variants are splice variants of a nucleic acid encodingbHLH transcription factor represented by any of SEQ ID NO 213, SEQ ID NO215, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO 225, SEQ ID NO: 297, SEQID NO: 299, and SEQ ID NO: 301, or splice variants encoding orthologuesor paralogues of any of the aforementioned SEQ ID NOs. Further preferredare splice variants of nucleic acids represented by any one of SEQ IDNO: 212, SEQ ID NO: 214, SEQ ID NO: 216, SEQ ID NO: 218, SEQ ID NO: 220,SEQ ID NO: 222, SEQ ID NO: 224, SEQ ID NO: 226, SEQ ID NO: 228, SEQ IDNO: 296, SEQ ID NO: 298, and SEQ ID NO: 300. Most preferred is a splicevariant of a nucleic acid as represented by SEQ ID NO: 212.

Another nucleic acid variant useful in performing the methods of theinvention is an allelic variant of a nucleic acid encoding a bHLHtranscription factor as defined hereinabove.

According to the present invention, there is provided a method forenhancing yield-related traits in plants, comprising introducing andexpressing in a plant an allelic variant of a nucleic acid encoding abHLH transcription factor represented by any of SEQ ID NO 213, SEQ ID NO215, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO 225, SEQ ID NO: 297, SEQID NO: 299, and SEQ ID NO: 301, or comprising introducing and expressingin a plant an allelic variant of a nucleic acid encoding an orthologue,paralogue or homologue of any of the aforementioned SEQ ID NOs.

The allelic variant may be an allelic variant of a nucleic acid encodinga bHLH transcription factor represented by any of SEQ ID NO 213, SEQ IDNO 215, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO 225, SEQ ID NO: 297,SEQ ID NO: 299, and SEQ ID NO: 301, or an allelic variants of a nucleicacid encoding orthologues or paralogues of any of the aforementioned SEQID NOs. Further preferred are allelic variants of nucleic acidsrepresented by any one of SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO:216, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO: 224, SEQID NO: 226 and SEQ ID NO: 228, SEQ ID NO: 296, SEQ ID NO: 298, SEQ IDNO: 300. Most preferred is an allelic variant of a nucleic acid asrepresented by SEQ ID NO: 212.

A further nucleic acid variant useful in the methods of the invention isa nucleic acid variant obtained by gene shuffling. Gene shuffling ordirected evolution may also be used to generate variants of nucleicacids encoding bHLH transcription factors as defined above.

According to the present invention, there is provided a method forenhancing yield-related traits in plants, comprising introducing andexpressing in a plant a variant of a nucleic acid encoding a bHLHtranscription factor represented by any of SEQ ID NO 213, SEQ ID NO 215,SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO 225, SEQ ID NO: 297, SEQ IDNO: 299, SEQ and ID NO: 301, or comprising introducing and expressing ina variant of a nucleic acid encoding an orthologue, paralogue orhomologue of any of the aforementioned SEQ ID NOs, which nucleic acid isobtained by gene shuffling.

Furthermore, nucleic acid variants may also be obtained by site-directedmutagenesis. Several methods are available to achieve site-directedmutagenesis, the most common being PCR based methods (current protocolsin molecular biology. Wiley Eds.).

Also useful in the methods of the invention are nucleic acids encodinghomologues of any one of the amino acids represented by SEQ ID NO 213,SEQ ID NO 215, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO 225, SEQ ID NO:297, SEQ ID NO: 299, SEQ ID NO: 301 or orthologues or paralogues of anyof the aforementioned SEQ ID NOs.

Also useful in the methods of the invention are nucleic acids encodingderivatives of any one of the amino acids represented by SEQ ID NO 213,SEQ ID NO 215, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO 225, SEQ ID NO:297, SEQ ID NO: 299, SEQ ID NO: 301 or orthologues or paralogues of anyof the aforementioned SEQ ID NOs. Derivatives of SEQ ID NO: 215, SEQ IDNO: 217, SEQ ID NO: 219, SEQ ID NO: 221, SEQ ID NO: 223, SEQ ID NO: 225,SEQ ID NO: 227 and SEQ ID NO: 229, SEQ ID NO: 297, SEQ ID NO: 299, SEQID NO: 301 are further examples which may be suitable for use in themethods of the invention

Furthermore, bHLH transcription factors (at least in their native form)typically have DNA-binding activity and an activation domain. A personskilled in the art may easily determine the presence of an activationdomain and DNA-binding activity using routine tools and techniques.

Nucleic acids encoding bHLH transcription factors may be derived fromany natural or artificial source. The nucleic acid may be modified fromits native form in composition and/or genomic environment throughdeliberate human manipulation. Preferably the bHLH transcriptionfactor-encoding nucleic acid is from a plant, further preferably from amonocot, more preferably from the Poaceae family, most preferably thenucleic acid is from Oryza sativa.

Any reference herein to a bHLH transcription factor is therefore takento mean a bHLH transcription factor as defined above. Any nucleic acidencoding such a bHLH transcription factor is suitable for use inperforming the methods of the invention.

The present invention also encompasses plants or parts thereof(including plant cells) obtainable by the methods according to thepresent invention. The plants or parts thereof comprise a nucleic acidtransgene encoding a bHLH transcription factor as defined above.

The invention also provides genetic constructs and vectors to facilitateintroduction and/or expression of the nucleic acid sequences useful inthe methods according to the invention, in a plant.

Therefore, there is provided a gene construct comprising:

-   -   (i) Any nucleic acid encoding a bHLH-type transcription factor        as defined hereinabove;    -   (ii) One or more control sequences operably liked to the nucleic        acid of (i).

Constructs useful in the methods according to the present invention maybe constructed using recombinant DNA technology well known to personsskilled in the art. The gene constructs may be inserted into vectors,which may be commercially available, suitable for transforming intoplants and suitable for expression of the gene of interest in thetransformed cells. The invention therefore provides use of a geneconstruct as defined hereinabove in the methods of the invention.

Plants are transformed with a vector comprising the sequence of interest(i.e., a nucleic acid encoding a bHLH-type transcription factor). Theskilled artisan is well aware of the genetic elements that must bepresent on the vector in order to successfully transform, select andpropagate host cells containing the sequence of interest. The sequenceof interest is operably linked to one or more control sequences (atleast to a promoter).

Advantageously, any type of promoter, whether natural or synthetic, maybe used to drive expression of the nucleic acid sequence. The promotermay be an inducible promoter, i.e. having induced or increasedtranscription initiation in response to a developmental, chemical,environmental or physical stimulus. The promoter may be atissue-specific promoter, i.e. one that is capable of preferentiallyinitiating transcription in certain tissues, such as the leaves, roots,seed tissue etc.

According to one preferred feature of the invention, the nucleic acidencoding a bHLH-type transcription factor is operably linked to aconstitutive promoter. A constitutive promoter is transcriptionallyactive during most but not necessarily all phases of its growth anddevelopment and is substantially ubiquitously expressed. Theconstitutive promoter is preferably a GOS2 promoter, more preferably theconstitutive promoter is a rice GOS2 promoter, further preferably theconstitutive promoter is represented by a nucleic acid sequencesubstantially similar to SEQ ID NO: 230 or SEQ ID NO: 233, mostpreferably the constitutive promoter is as represented by SEQ ID NO:233.

It should be clear that the applicability of the present invention isnot restricted to the bHLH transcription factor-encoding nucleic acidrepresented by SEQ ID NO: 212, nor is the applicability of the inventionrestricted to expression of a such a bHLH transcription factor-encodingnucleic acid when driven by a GOS2 promoter. Examples of otherconstitutive promoters which may also be used perform the methods of theinvention are shown in the definitions section.

Optionally, one or more terminator sequences (also a control sequence)may be used in the construct introduced into a plant. Additionalregulatory elements may include transcriptional as well as translationalenhancers. Those skilled in the art will be aware of terminator andenhancer sequences that may be suitable for use in performing theinvention. Such sequences would be known or may readily be obtained by aperson skilled in the art.

The genetic constructs of the invention may further include an origin ofreplication sequence that is required for maintenance and/or replicationin a specific cell type. One example is when a genetic construct isrequired to be maintained in a bacterial cell as an episomal geneticelement (e.g. plasmid or cosmid molecule). Preferred origins ofreplication include, but are not limited to, the f1-ori and colE1. Othercontrol sequences (besides promoter, enhancer, silencer, intronsequences, 3′UTR and/or 5′UTR regions) may be protein and/or RNAstabilizing elements. The genetic construct may optionally comprise aselectable marker gene.

The invention also provides a method for the production of transgenicplants having enhanced yield-related traits relative to control plants,comprising introduction and expression in a plant of any nucleic acidencoding a bHLH-type transcription factor polypeptide as definedhereinabove.

More specifically, the present invention provides a method for theproduction of transgenic plants having enhanced yield-related traits,which method comprises:

-   -   (i) introducing and expressing a nucleic acid encoding a        bHLH-type transcription factor (as defined herein) in a plant        cell; and    -   (ii) cultivating the plant cell under conditions promoting plant        growth and development.

The nucleic acid may be introduced directly into a plant cell or intothe plant itself (including introduction into a tissue, organ or anyother part of a plant). According to a preferred feature of the presentinvention, the nucleic acid is preferably introduced into a plant bytransformation.

Generally after transformation, plant cells or cell groupings areselected for the presence of one or more markers which are encoded byplant-expressible genes co-transferred with the gene of interest,following which the transformed material is regenerated into a wholeplant.

Following DNA transfer and regeneration, putatively transformed plantsmay be evaluated, for instance using Southern analysis, for the presenceof the gene of interest, copy number and/or genomic organisation.Alternatively or additionally, expression levels of the newly introducedDNA may be monitored using Northern and/or Western analysis, orquantitative PCR, all techniques being well known to persons havingordinary skill in the art.

The generated transformed plants may be propagated by a variety ofmeans, such as by clonal propagation or classical breeding techniques.For example, a first generation (or T1) transformed plant may be selfedto give homozygous second generation (or T2) transformants, and the T2plants further propagated through classical breeding techniques.

The generated transformed organisms may take a variety of forms. Forexample, they may be chimeras of transformed cells and non-transformedcells; clonal transformants (e.g., all cells transformed to contain theexpression cassette); grafts of transformed and untransformed tissues(e.g., in plants, a transformed rootstock grafted to an untransformedscion).

The present invention clearly extends to any plant cell or plantproduced by any of the methods described herein, and to all plant partsand propagules thereof. The present invention extends further toencompass the progeny of a primary transformed or transfected cell,tissue, organ or whole plant that has been produced by any of theaforementioned methods, the only requirement being that progeny exhibitthe same genotypic and/or phenotypic characteristic(s) as those producedby the parent in the methods according to the invention.

The invention also includes host cells containing an isolated nucleicacid encoding a bHLH transcription factor as defined hereinabove.Preferred host cells according to the invention are plant cells.

The invention also extends to harvestable parts of a plant such as, butnot limited to seeds, leaves, fruits, flowers, stems, rhizomes, tubersand bulbs. The invention furthermore relates to products derived,preferably directly derived, from a harvestable part of such a plant,such as dry pellets or powders, oil, fat and fatty acids, starch orproteins.

According to a preferred feature of the invention, the modulatedexpression is increased expression.

As mentioned above, a preferred method for modulating (preferably,increasing) expression of a nucleic acid encoding a bHLH transcriptionfactor is by introducing and expressing in a plant a nucleic acidencoding a bHLH transcription factor; however the effects of performingthe method, i.e. enhancing yield-related traits may also be achievedusing other well known techniques. A description of some of thesetechniques will now follow.

One such technique is T-DNA activation tagging. The resulting transgenicplants show dominant phenotypes due to modified expression of genesclose to the introduced promoter. The effects of the invention may alsobe reproduced using the technique of TILLING (Targeted Induced LocalLesions In Genomes). The effects of the invention may also be reproducedusing homologous recombination.

Reference herein to the term enhanced yield-related traits is taken tomean an increase in biomass (weight) of one or more parts of a plant,which may include aboveground (harvestable) parts and/or (harvestable)parts below ground.

In particular, such harvestable parts are seeds, and performance of themethods of the invention results in plants having increased seed yieldrelative to the seed yield of control plants.

Taking corn as an example, a yield increase may be manifested as one ormore of the following: increase in the number of plants established perhectare or acre, an increase in the number of ears per plant, anincrease in the number of rows, number of kernels per row, kernelweight, thousand kernel weight, ear length/diameter, increase in theseed filling rate (which is the number of filled seeds divided by thetotal number of seeds and multiplied by 100), among others. Taking riceas an example, a yield increase may manifest itself as an increase inone or more of the following: number of plants per hectare or acre,number of panicles per plant, number of spikelets per panicle, number offlowers (florets) per panicle (which is expressed as a ratio of thenumber of filled seeds over the number of primary panicles), increase inthe seed filling rate (which is the number of filled seeds divided bythe total number of seeds and multiplied by 100), increase in thousandkernel weight, among others.

Since the transgenic plants according to the present invention haveincreased yield, it is likely that these plants exhibit an increasedgrowth rate (during at least part of their life cycle), relative to thegrowth rate of corresponding wild type plants at a corresponding stagein their life cycle. The increased growth rate may be specific to one ormore parts of a plant (including seeds), or may be throughoutsubstantially the whole plant. A plant having an increased growth ratemay even exhibit early flowering. The increase in growth rate may takeplace at one or more stages in the life cycle of a plant or duringsubstantially the whole plant life cycle. Increased growth rate duringthe early stages in the life cycle of a plant may reflect enhancedvigour (increased seedling vigor at emergence). The increase in growthrate may alter the harvest cycle of a plant allowing plants to be sownlater and/or harvested sooner than would otherwise be possible. If thegrowth rate is sufficiently increased, it may allow for the furthersowing of seeds of the same plant species (for example sowing andharvesting of rice plants followed by sowing and harvesting of furtherrice plants all within one conventional growing period). Similarly, ifthe growth rate is sufficiently increased, it may allow for the furthersowing of seeds of different plants species (for example the sowing andharvesting of corn plants followed by, for example, the sowing andoptional harvesting of soy bean, potato or any other suitable plant).Harvesting additional times from the same rootstock in the case of somecrop plants may also be possible. Altering the harvest cycle of a plantmay lead to an increase in annual biomass production per acre (due to anincrease in the number of times (say in a year) that any particularplant may be grown and harvested). An increase in growth rate may alsoallow for the cultivation of transgenic plants in a wider geographicalarea than their wild-type counterparts, since the territoriallimitations for growing a crop are often determined by adverseenvironmental conditions either at the time of planting (early season)or at the time of harvesting (late season). Such adverse conditions maybe avoided if the harvest cycle is shortened. The growth rate may bedetermined by deriving various parameters from growth curves, suchparameters may be: T-Mid (the time taken for plants to reach 50% oftheir maximal size) and T-90 (time taken for plants to reach 90% oftheir maximal size), amongst others.

An increase in yield and/or growth rate occurs whether the plant isunder non-stress conditions or whether the plant is exposed to variousstresses compared to control plants. Plants typically respond toexposure to stress by growing more slowly. In conditions of severestress, the plant may even stop growing altogether. Mild stress on theother hand is defined herein as being any stress to which a plant isexposed which does not result in the plant ceasing to grow altogetherwithout the capacity to resume growth. Mild stress in the sense of theinvention leads to a reduction in the growth of the stressed plants ofless than 40%, 35% or 30%, preferably less than 25%, 20% or 15%, morepreferably less than 14%, 13%, 12%, 11% or 10% or less in comparison tothe control plant under non-stress conditions. Due to advances inagricultural practices (irrigation, fertilization, pesticide treatments)severe stresses are not often encountered in cultivated crop plants. Asa consequence, the compromised growth induced by mild stress is often anundesirable feature for agriculture. Mild stresses are the everydaybiotic and/or abiotic (environmental) stresses to which a plant isexposed. Abiotic stresses may be due to drought or excess water,anaerobic stress, salt stress, chemical toxicity, oxidative stress andhot, cold or freezing temperatures. The abiotic stress may be an osmoticstress caused by a water stress (particularly due to drought), saltstress, oxidative stress or an ionic stress. Biotic stresses aretypically those stresses caused by pathogens, such as bacteria, viruses,fungi and insects.

In particular, the methods of the present invention may be performedunder non-stress conditions or under conditions of mild drought to giveplants having increased yield relative to control plants. As reported inWang et al. (Planta (2003) 218: 1-14), abiotic stress leads to a seriesof morphological, physiological, biochemical and molecular changes thatadversely affect plant growth and productivity. Drought, salinity,extreme temperatures and oxidative stress are known to be interconnectedand may induce growth and cellular damage through similar mechanisms.Rabbani et al. (Plant Physiol (2003) 133: 1755-1767) describes aparticularly high degree of “cross talk” between drought stress andhigh-salinity stress. For example, drought and/or salinisation aremanifested primarily as osmotic stress, resulting in the disruption ofhomeostasis and ion distribution in the cell. Oxidative stress, whichfrequently accompanies high or low temperature, salinity or droughtstress, may cause denaturing of functional and structural proteins. As aconsequence, these diverse environmental stresses often activate similarcell signaling pathways and cellular responses, such as the productionof stress proteins, up-regulation of anti-oxidants, accumulation ofcompatible solutes and growth arrest. The term “non-stress” conditionsas used herein are those environmental conditions that allow optimalgrowth of plants. Persons skilled in the art are aware of normal soilconditions and climatic conditions for a given location.

Performance of the methods of the invention gives plants grown undernon-stress conditions or under mild drought conditions increased yieldrelative to control plants grown under comparable conditions. Therefore,according to the present invention, there is provided a method forincreasing yield in plants grown under non-stress conditions or undermild drought conditions, which method comprises increasing expression ina plant of a nucleic acid encoding a bHLH transcription factor.

Performance of the methods of the invention gives plants grown underconditions of nutrient deficiency, particularly under conditions ofnitrogen deficiency, increased yield relative to control plants grownunder comparable conditions. Therefore, according to the presentinvention, there is provided a method for increasing yield in plantsgrown under conditions of nutrient deficiency, which method comprisesincreasing expression in a plant of a nucleic acid encoding a MADS15polypeptide. Nutrient deficiency may result from a lack or excess ofnutrients such as nitrogen, phosphates and other phosphorous-containingcompounds, potassium, calcium, cadmium, magnesium, manganese, iron andboron, amongst others.

According to a preferred feature of the present invention, performanceof the methods of the invention gives plants having an increased growthrate relative to control plants, particularly during the early stages ofplant development (typically three weeks post germination in the case ofrice and maize, but this will vary from species to species) leading toearly vigour. Therefore, according to the present invention, there isprovided a method for increasing the growth rate of plants, which methodcomprises modulating, preferably increasing, expression in a plant of anucleic acid encoding a bHLH transcription factor. The present inventiontherefore also provides a method for obtaining plants having earlyvigour relative to control plants, which method comprises modulating,preferably increasing, expression in a plant of a nucleic acid encodinga bHLH transcription factor.

Early vigour may also result from or be manifested as increased plantfitness relative to control plants due to, for example, the plants beingbetter adapted to their environment (i.e. being more able to cope withvarious abiotic or biotic stress factors). Plants having early vigouralso show better establishment of the crop (with the crop growing inuniform manner, i.e. with the majority of plants reaching the variousstages of development at substantially the same time), and show bettergrowth and often better yield. Early vigour may be determined bymeasuring various factors, such as seedling growth rate, thousand kernelweight, percentage germination, percentage emergence, seedling height,root length and shoot biomass and many more.

The methods of the invention are advantageously applicable to any plant.

Plants that are particularly useful in the methods of the inventioninclude all plants which belong to the superfamily Viridiplantae, inparticular monocotyledonous and dicotyledonous plants including fodderor forage legumes, ornamental plants, food crops, trees or shrubs.According to a preferred embodiment of the present invention, the plantis a crop plant. Examples of crop plants include soybean, sunflower,canola, alfalfa, rapeseed, cotton, tomato, potato and tobacco. Furtherpreferably, the plant is a monocotyledonous plant. Examples ofmonocotyledonous plants include sugarcane. More preferably the plant isa cereal. Examples of cereals include rice, maize, wheat, barley,millet, rye, triticale, sorghum and oats. Plants in which early vigouris a particularly desirably trait include rice, maize, wheat, sunflower,sorghum.

The present invention also encompasses use of nucleic acids encodingbHLH transcription factors and use of bHLH transcription factorpolypeptides in enhancing yield-related traits.

Nucleic acids encoding bHLH transcription factor polypeptides, or bHLHtranscription factors themselves, may find use in breeding programmes inwhich a DNA marker is identified which may be genetically linked to abHLH transcription factor-encoding gene. The nucleic acids/genes, or thebHLH transcription factors themselves may be used to define a molecularmarker. This DNA or protein marker may then be used in breedingprogrammes to select plants having increased yield as definedhereinabove in the methods of the invention.

Allelic variants of a bHLH transcription factor-encoding acid/gene mayalso find use in marker-assisted breeding programmes. Such breedingprogrammes sometimes require introduction of allelic variation bymutagenic treatment of the plants, using for example EMS mutagenesis;alternatively, the programme may start with a collection of allelicvariants of so called “natural” origin caused unintentionally.Identification of allelic variants then takes place, for example, byPCR. This is followed by a step for selection of superior allelicvariants of the sequence in question and which give increased yield.Selection is typically carried out by monitoring growth performance ofplants containing different allelic variants of the sequence inquestion. Growth performance may be monitored in a greenhouse or in thefield. Further optional steps include crossing plants in which thesuperior allelic variant was identified with another plant. This couldbe used, for example, to make a combination of interesting phenotypicfeatures.

A nucleic acid encoding a bHLH transcription factor may also be used asprobes for genetically and physically mapping the genes that they are apart of, and as markers for traits linked to those genes. Suchinformation may be useful in plant breeding in order to develop lineswith desired phenotypes. Such use of bHLH transcription factor encodingnucleic acids requires only a nucleic acid sequence of at least 15nucleotides in length. The bHLH transcription factor encoding nucleicacids may be used as restriction fragment length polymorphism (RFLP)markers. Southern blots (Sambrook J, Fritsch E F and Maniatis T (1989)Molecular Cloning, A Laboratory Manual) of restriction-digested plantgenomic DNA may be probed with the bHLH transcription factor encodingnucleic acids. The resulting banding patterns may then be subjected togenetic analyses using computer programs such as MapMaker (Lander et al.(1987) Genomics 1: 174-181) in order to construct a genetic map. Inaddition, the nucleic acids may be used to probe Southern blotscontaining restriction endonuclease-treated genomic DNAs of a set ofindividuals representing parent and progeny of a defined genetic cross.Segregation of the DNA polymorphisms is noted and used to calculate theposition of the bHLH transcription factor encoding nucleic acid in thegenetic map previously obtained using this population (Botstein et al.(1980) Am. J. Hum. Genet. 32:314-331).

The production and use of plant gene-derived probes for use in geneticmapping is described in Bernatzky and Tanksley (1986) Plant Mol. Biol.Reporter 4: 37-41. Numerous publications describe genetic mapping ofspecific cDNA clones using the methodology outlined above or variationsthereof. For example, F2 intercross populations, backcross populations,randomly mated populations, near isogenic lines, and other sets ofindividuals may be used for mapping. Such methodologies are well knownto those skilled in the art.

The nucleic acid probes may also be used for physical mapping (i.e.,placement of sequences on physical maps; see Hoheisel et al. In:Non-mammalian Genomic Analysis: A Practical Guide, Academic press 1996,pp. 319-346, and references cited therein).

In another embodiment, the nucleic acid probes may be used in directfluorescence in situ hybridisation (FISH) mapping (Trask (1991) TrendsGenet. 7:149-154). Although current methods of FISH mapping favor use oflarge clones (several kb to several hundred kb; see Laan et al. (1995)Genome Res. 5:13-20), improvements in sensitivity may allow performanceof FISH mapping using shorter probes.

A variety of nucleic acid amplification-based methods for genetic andphysical mapping may be carried out using the nucleic acids. Examplesinclude allele-specific amplification (Kazazian (1989) J. Lab. Clin.Med. 11:95-96), polymorphism of PCR-amplified fragments (CAPS; Sheffieldet al. (1993) Genomics 16:325-332), allele-specific ligation (Landegrenet al. (1988) Science 241:1077-1080), nucleotide extension reactions(Sokolov (1990) Nucleic Acid Res. 18:3671), Radiation Hybrid Mapping(Walter et al. (1997) Nat. Genet. 7:22-28) and Happy Mapping (Dear andCook (1989) Nucleic Acid Res. 17:6795-6807). For these methods, thesequence of a nucleic acid is used to design and produce primer pairsfor use in the amplification reaction or in primer extension reactions.The design of such primers is well known to those skilled in the art. Inmethods employing PCR-based genetic mapping, it may be necessary toidentify DNA sequence differences between the parents of the mappingcross in the region corresponding to the instant nucleic acid sequence.This, however, is generally not necessary for mapping methods.

The methods according to the present invention result in plants havingenhanced yield-related traits, as described hereinbefore. These traitsmay also be combined with other economically advantageous traits, suchas further yield-enhancing traits, tolerance to other abiotic and bioticstresses, traits modifying various architectural features and/orbiochemical and/or physiological features.

Detailed Description of SPL15

Surprisingly, it has now been found that increasing expression in aplant of a nucleic acid encoding an SPL15 transcription factorpolypeptide gives plants having enhanced yield related traits,particularly increased yield, relative to control plants. Therefore, theinvention provides a method for enhancing yield related traits inparticular for increasing yield in plants relative to control plants,comprising increasing expression in a plant of a nucleic acid encodingan SPL15 transcription factor polypeptide.

A preferred method for increasing expression of a nucleic acid encodinga SPL15 transcription factor polypeptide is by introducing andexpressing in a plant a nucleic acid encoding a SPL15 transcriptionfactor polypeptide.

The term “SPL15 transcription factor polypeptide” as defined hereinrefers to a polypeptide comprising from N-terminal to C-terminal: (i)Motif 1 as represented by SEQ ID NO: 276; and (ii) in increasing orderof preference at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% sequenceidentity to the SPL DBD represented by SEQ ID NO: 277; and (iii) Motif 2as represented by SEQ ID NO: 278.

The most conserved amino acids within Motif 1 are XLXFGXXXYFX, andwithin Motif 2 DSXXALSLLSX (where X is a specified subset of amino acidsdiffering for each position, as presented in SEQ ID NO: 276 and SEQ IDNO: 277). Within Motif 1 and Motif 2, are allowed one or moreconservative change at any position, and/or one, two or threenon-conservative change(s) at any position.

Additionally, the SPL15 transcription factor polypeptide may compriseany one or both of the following: (a) a G/S rich stretch preceding theSPL DBD; and (b) the W(S/T)L tripeptide at the C-terminal end of thepolypeptide.

An example of an SPL15 transcription polypeptide as defined hereinabovecomprising from N-terminal to C-terminal: (i) Motif 1 as represented bySEQ ID NO: 276; and (ii) in increasing order of preference at least 65%,70%, 75%, 80%, 85%, 90%, 95% or 98% sequence identity to the SPL DBDrepresented by SEQ ID NO: 277; and (iii) Motif 2 as represented by SEQID NO: 278; and additionally comprising: (a) a G/S rich stretchpreceding the SPL DBD; and (b) the W(S/T)L tripeptide at the C-terminalend of the polypeptide, is represented as in SEQ ID NO: 235. Furthersuch examples are represented by any one of SEQ ID NO: 237, SEQ ID NO:239, SEQ ID NO: 241, SEQ ID NO: 243, SEQ ID NO: 245, SEQ ID NO: 247, SEQID NO: 249, SEQ ID NO: 251, SEQ ID NO: 253, SEQ ID NO: 255, SEQ ID NO:257, SEQ ID NO: 259, SEQ ID NO: 261, SEQ ID NO: 263, SEQ ID NO: 283, SEQID NO: 285, SEQ ID NO: 287 or orthologues or paralogues of any of theaforementioned SEQ ID NOs. The invention is illustrated by transformingplants with the Arabidopsis thaliana sequence represented by SEQ ID NO:234, encoding the polypeptide of SEQ ID NO: 235. SEQ ID NO: 237 fromArabidopsis thaliana (encoded by SEQ ID NO: 236) is a paralogue of thepolypeptide of SEQ ID NO: 235. SEQ ID NO: 239 (encoded by SEQ ID NO:238, from Aquilegia formosa x Aquilegia pubescens), SEQ ID NO: 241(encoded by SEQ ID NO: 240, from Gossypium hirsutum), SEQ ID NO: 243(encoded by SEQ ID NO: 242, from Ipomoea nil), SEQ ID NO: 245 (encodedby SEQ ID NO: 244, from Lactuca sativa), SEQ ID NO: 247 (encoded by SEQID NO: 246, from Malus domestica), SEQ ID NO: 249 (encoded by SEQ ID NO:248, from Medicago truncatula), SEQ ID NO: 251 (encoded by SEQ ID NO:250, from Nicotiana bentamiana), SEQ ID NO: 253 (encoded by SEQ ID NO:252, from Oryza sativa), SEQ ID NO: 255 (encoded by SEQ ID NO: 254, fromOryza sativa), SEQ ID NO: 257 (encoded by SEQ ID NO: 256, from Solanumtuberosum SEQ ID NO: 259 (encoded by SEQ ID NO: 258, from Vitisvinifera), SEQ ID NO: 261 (encoded by SEQ ID NO: 260, from Zea mays),SEQ ID NO: 263 (encoded by SEQ ID NO: 262, Zea mays), SEQ ID NO: 283(encoded by SEQ ID NO: 282, Brassica rapa), SEQ ID NO: 285 (encoded bySEQ ID NO: 284, Glycine max), and SEQ ID NO: 287 (encoded by SEQ ID NO:286, Populus tremuloides) are orthologues of the polypeptide of SEQ IDNO: 235.

Orthologues and paralogues may easily be found by performing a so-calledreciprocal blast search. This may be done by a first BLAST involvingBLASTing a query sequence (for example, SEQ ID NO: 234 or SEQ ID NO:235) against any sequence database, such as the publicly available NCBIdatabase. BLASTN or TBLASTX (using standard default values) may be usedwhen starting from a nucleotide sequence and BLASTP or TBLASTN (usingstandard default values) may be used when starting from a polypeptidesequence. The BLAST results may optionally be filtered. The full-lengthsequences of either the filtered results or non-filtered results arethen BLASTed back (second BLAST) against sequences from the organismfrom which the query sequence is derived (where the query sequence isSEQ ID NO: 234 or SEQ ID NO: 235, the second BLAST would therefore beagainst Arabidopsis sequences). The results of the first and secondBLASTs are then compared. A paralogue is identified if a high-rankinghit from the first BLAST is from the same species as from which thequery sequence is derived, a BLAST back then ideally results in thequery sequence as highest hit (besides itself); an orthologue isidentified if a high-ranking hit in the first BLAST is not from the samespecies as from which the query sequence is derived and preferablyresults upon BLAST back in the query sequence amongst the highest hits.High-ranking hits are those having a low E-value. The lower the E-value,the more significant the score (or in other words the lower the chancethat the hit was found by chance). Computation of the E-value is wellknown in the art. In addition to E-values, comparisons are also scoredby percentage identity. Percentage identity refers to the number ofidentical nucleotides (or amino acids) between the two compared nucleicacid (or polypeptide) sequences over a particular length. An exampledetailing the identification of orthologues and paralogues is given inExample 41. In the case of large families, ClustalW may be used,followed by a neighbour joining tree, to help visualize clustering ofrelated genes and to identify orthologues and paralogues. In FIG. 22,the SPL15 transcription factor polypeptide paralogues and orthologuescluster together.

The polypeptides represented by any one of SEQ ID NO: 237, SEQ ID NO:239, SEQ ID NO: 241, SEQ ID NO: 243, SEQ ID NO: 245, SEQ ID NO: 247, SEQID NO: 249, SEQ ID NO: 251, SEQ ID NO: 253, SEQ ID NO: 255, SEQ ID NO:257, SEQ ID NO: 259, SEQ ID NO: 261, SEQ ID NO: 263, SEQ ID NO: 283, SEQID NO: 285, SEQ ID NO: 287, or orthologues or paralogues of any of theaforementioned SEQ ID NOs, all comprise an SPL DBD having, in increasingorder of preference, at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98%sequence identity to the SPL15 DBD represented by SEQ ID NO: 277.

SPL DBDs are well known in the art and may readily be identified bypersons skilled in the art. A SPL DBD may be identified using methodsfor the alignment of sequences for comparison. Methods for the alignmentof sequences for comparison include GAP, BESTFIT, BLAST, FASTA andTFASTA. GAP uses the algorithm of Needleman and Wunsch ((1970) J MolBiol 48: 443-453) to find the alignment of two complete sequences thatmaximizes the number of matches and minimizes the number of gaps. TheBLAST algorithm (Altschul et al. (1990) J Mol Biol 215: 403-10)calculates percent sequence identity and performs a statistical analysisof the similarity between the two sequences. The software for performingBLAST analysis is publicly available through the National Centre forBiotechnology Information. Homologues may readily be identified using,for example, the ClustalW multiple sequence alignment algorithm (version1.83) available at GenomeNet service at the Kyoto UniversityBioinformatics Center, with the default pairwise alignment parameters,and a scoring method in percentage. Minor manual editing may beperformed to optimise alignment between conserved motifs, as would beapparent to a person skilled in the art. The alignment shown in FIGS. 23and 24 highlight the SPL DBD in SPL15 transcription factor polypeptides.Preferably, SPL15 transcription factor polypeptides useful in themethods of the invention comprise an SPL DBD having, in increasing orderof preference, at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98%sequence identity to the SPL DBD represented by SEQ ID NO: 277.

In some instances, default parameters may be adjusted to modify thestringency of the search. For example using BLAST, the statisticalsignificance threshold (called “expect” value) for reporting matchesagainst database sequences may be increased to show less stringentmatches. In this way, short nearly exact matches may be identified.Motif 1 as represented by SEQ ID NO: 276 and Motif 2 as represented bySEQ ID NO: 278 both comprised in the SPL15 transcription factorpolypeptides useful in the methods of the invention may be identifiedthis way (FIG. 24). Within Motif 1 and Motif 2, are allowed one or moreconservative change at any position, and/or one, two or threenon-conservative change(s) at any position. The W(S/T)L tripeptide atthe C-terminal end of the polypeptide may likewise be identified (FIG.24).

Special databases also exist for the identification of domains. The SPLDBD in a SPL15 transcription factor polypeptide may be identified using,for example, SMART (Schultz et al. (1998) Proc. Natl. Acad. Sci. USA 95,5857-5864; Letunic et al. (2002) Nucleic Acids Res 30, 242-244; hostedby the EMBL at Heidelberg, Germany), InterPro (Mulder et al., (2003)Nucl. Acids. Res. 31, 315-318; hosted by the European BioinformaticsInstitute (EBI) in the United Kingdom), Prosite (Bucher and Bairoch(1994), A generalized profile syntax for biomolecular sequences motifsand its function in automatic sequence interpretation. (In) ISMB-94;Proceedings 2nd International Conference on Intelligent Systems forMolecular Biology. Altman R., Brutlag D., Karp P., Lathrop R., SearlsD., Eds., pp 53-61, AAAIPress, Menlo Park; Hulo et al., Nucl. Acids.Res. 32: D134-D137, (2004), The ExPASy proteomics server is provided asa service to the scientific community (hosted by the Swiss Institute ofBioinformatics (SIB) in Switzerland) or Pfam (Bateman et al., NucleicAcids Research 30(1): 276-280 (2002), hosted by the Sanger Institute inthe United Kingdom). The SPL DBD in the InterPro database is designatedIPR004333, PF03110 in the Pfam database and PS51141 in the PROSITEdatabase.

Furthermore, the presence of G/S rich stretch preceding the SPL DBD mayalso readily be identified (FIG. 24). Primary amino acid composition (in%) to determine if a polypeptide domain is rich in specific amino acidsmay be calculated using software programs from the ExPASy server, inparticular the ProtParam tool (Gasteiger E et al. (2003) ExPASy: theproteomics server for in-depth protein knowledge and analysis. NucleicAcids Res 31:3784-3788). The composition of the protein of interest maythen be compared to the average amino acid composition (in %) in theSwiss-Prot Protein Sequence data bank. Within this databank, the averageGly (G) and Ser (E) content are both of 6.9% (adding up to 13.8%). As anexample, the G/S rich stretch preceding the SPL DBD of SEQ ID NO: 235contains 14.5% of G and 25.5% of S (adding up to 40%). As definedherein, a G/S rich stretch has a combined G and S content (in % terms)above that found in the average amino acid composition (in % terms) ofthe proteins in the Swiss-Prot Protein Sequence. Both G and S belong tothe category of very small amino acids.

The nucleic acid encoding the polypeptides represented by any one of SEQID NO: 235, SEQ ID NO: 237, SEQ ID NO: 239, SEQ ID NO: 241, SEQ ID NO:243, SEQ ID NO: 245, SEQ ID NO: 247, SEQ ID NO: 249, SEQ ID NO: 251, SEQID NO: 253, SEQ ID NO: 255, SEQ ID NO: 257, SEQ ID NO: 259, SEQ ID NO:261, SEQ ID NO: 263, SEQ ID NO: 283, SEQ ID NO: 285, SEQ ID NO: 287, ororthologues or paralogues of any of the aforementioned SEQ ID NOs, neednot be full-length nucleic acids, since performance of the methods ofthe invention does not rely on the use of full length nucleic acidsequences. Furthermore, examples of nucleic acids suitable for use inperforming the methods of the invention include but are not limited tothose represented by any one of: SEQ ID NO: 234, SEQ ID NO: 236, SEQ IDNO: 238, SEQ ID NO: 240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO: 246,SEQ ID NO: 248, SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO: 254, SEQ IDNO: 256, SEQ ID NO: 258, SEQ ID NO: 260, SEQ ID NO: 262, SEQ ID NO: 282,SEQ ID NO: 284 and SEQ ID NO: 286. Nucleic acid variants may also beuseful in practising the methods of the invention. Examples of suchvariants include portions of nucleic acids, splice variants, allelicvariants either naturally occurring or obtained by DNA manipulation.

A portion may be prepared, for example, by making one or more deletionsto a nucleic acid encoding a SPL15 transcription factor polypeptide asdefined hereinabove. The portions may be used in isolated form or theymay be fused to other coding (or non coding) sequences in order to, forexample, produce a protein that combines several activities. When fusedto other coding sequences, the resultant polypeptide produced upontranslation may be bigger than that predicted for the SPL15transcription factor portion. Portions useful in the methods of theinvention, encode an SPL15 transcription factor polypeptide (asdescribed above) and having substantially the same biological activityas the SPL15 transcription factor polypeptide represented by any of SEQID NO: 235, SEQ ID NO: 237, SEQ ID NO: 239, SEQ ID NO: 241, SEQ ID NO:243, SEQ ID NO: 245, SEQ ID NO: 247, SEQ ID NO: 249, SEQ ID NO: 251, SEQID NO: 253, SEQ ID NO: 255, SEQ ID NO: 257, SEQ ID NO: 259, SEQ ID NO:261, SEQ ID NO: 263, SEQ ID NO: 283, SEQ ID NO: 285, SEQ ID NO: 287, ororthologues or paralogues of any of the aforementioned SEQ ID NOs.Examples of portions may include the nucleotides encoding Motif 1 asrepresented by SEQ ID NO: 276, in increasing order of preference atleast 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% sequence identity to theSPL DBD represented by SEQ ID NO: 277, and Motif 2 as represented by SEQID NO: 278. Portions may additionally include nucleotides encoding theG/S rich stretch or the W(S/T)L tripeptide (but not necessarily thenucleotides encoding the amino acid sequences between any of these). Theportion is typically at least 250 nucleotides in length, preferably atleast 500 nucleotides in length, more preferably at least 750nucleotides in length and most preferably at least 1000 nucleotides inlength. Preferably, the portion is a portion of a nucleic acid asrepresented by any one of SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO:238, SEQ ID NO: 240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO: 246, SEQID NO: 248, SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO: 254, SEQ ID NO:256, SEQ ID NO: 258, SEQ ID NO: 260, SEQ ID NO: 262, SEQ ID NO: 282, SEQID NO: 284 and SEQ ID NO: 286. Most preferably the portion is a portionof a nucleic acid as represented by SEQ ID NO: 234.

Another nucleic acid variant useful in the methods of the invention, isa nucleic acid capable of hybridising under reduced stringencyconditions, preferably under stringent conditions, with a nucleic acidencoding a SPL15 transcription factor polypeptide as definedhereinabove, or a with a portion as defined hereinabove.

Hybridising sequences useful in the methods of the invention, encode apolypeptide comprising from N-terminal to C-terminal: (i) Motif 1 asrepresented by SEQ ID NO: 276; and (ii) in increasing order ofpreference at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% sequenceidentity to the SPL DBD represented by SEQ ID NO: 277; and (iii) Motif 2as represented by SEQ ID NO: 278, and having substantially the samebiological activity as the SPL15 transcription factor polypeptidesrepresented by SEQ ID NO: 235, SEQ ID NO: 237, SEQ ID NO: 239, SEQ IDNO: 241, SEQ ID NO: 243, SEQ ID NO: 245, SEQ ID NO: 247, SEQ ID NO: 249,SEQ ID NO: 251, SEQ ID NO: 253, SEQ ID NO: 255, SEQ ID NO: 257, SEQ IDNO: 259, SEQ ID NO: 261, SEQ ID NO: 263, SEQ ID NO: 283, SEQ ID NO: 285,SEQ ID NO: 287, or orthologues or paralogues of any of theaforementioned SEQ ID NOs. The hybridising sequence is typically atleast 250 nucleotides in length, preferably at least 500 nucleotides inlength, more preferably at least 750 nucleotides in length and mostpreferably at least 1000 nucleotides in length. Preferably, thehybridising sequence is one that is capable of hybridising to any of thenucleic acids represented by SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO:238, SEQ ID NO: 240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO: 246, SEQID NO: 248, SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO: 254, SEQ ID NO:256, SEQ ID NO: 258, SEQ ID NO: 260, SEQ ID NO: 262, SEQ ID NO: 282, SEQID NO: 284 and SEQ ID NO: 286, or to a portion of any of theaforementioned sequences, a portion being as defined above. Mostpreferably the hybridising sequence is capable of hybridising to SEQ IDNO: 234, or to portions thereof.

Another nucleic acid variant useful in the methods of the invention is asplice variant encoding a SPL15 transcription factor polypeptide asdefined hereinabove.

Preferred splice variants are splice variants of a nucleic acid encodingSPL15 transcription factor polypeptide represented by any of SEQ ID NO:235, SEQ ID NO: 237, SEQ ID NO: 239, SEQ ID NO: 241, SEQ ID NO: 243, SEQID NO: 245, SEQ ID NO: 247, SEQ ID NO: 249, SEQ ID NO: 251, SEQ ID NO:253, SEQ ID NO: 255, SEQ ID NO: 257, SEQ ID NO: 259, SEQ ID NO: 261, SEQID NO: 263, SEQ ID NO: 283, SEQ ID NO: 285, or SEQ ID NO: 287, or splicevariants encoding orthologues or paralogues of any of the aforementionedSEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO: 240, SEQ IDNO: 242, SEQ ID NO: 244, SEQ ID NO: 246, SEQ ID NO: 248, SEQ ID NO: 250,SEQ ID NO: 252, SEQ ID NO: 254, SEQ ID NO: 256, SEQ ID NO: 258, SEQ IDNO: 260, SEQ ID NO: 262, SEQ ID NO: 282, SEQ ID NO: 284 and SEQ ID NO:286. Most preferred is a splice variant of a nucleic acid as representedby SEQ ID NO: 234.

Another nucleic acid variant useful in performing the methods of theinvention is an allelic variant of a nucleic acid encoding a SPL15transcription factor polypeptide as defined hereinabove.

The allelic variant may be an allelic variant of a nucleic acid encodinga SPL15 transcription factor polypeptide represented by any of SEQ IDNO: 235, SEQ ID NO: 237, SEQ ID NO: 239, SEQ ID NO: 241, SEQ ID NO: 243,SEQ ID NO: 245, SEQ ID NO: 247, SEQ ID NO: 249, SEQ ID NO: 251, SEQ IDNO: 253, SEQ ID NO: 255, SEQ ID NO: 257, SEQ ID NO: 259, SEQ ID NO: 261,SEQ ID NO: 263, SEQ ID NO: 283, SEQ ID NO: 285, or SEQ ID NO: 287, or anallelic variant of a nucleic acid encoding orthologues or paralogues ofany of the aforementioned SEQ ID NOs. Further preferred are allelicvariants of nucleic acids represented by any one of SEQ ID NO: 234, SEQID NO: 236, SEQ ID NO: 238, SEQ ID NO: 240, SEQ ID NO: 242, SEQ ID NO:244, SEQ ID NO: 246, SEQ ID NO: 248, SEQ ID NO: 250, SEQ ID NO: 252, SEQID NO: 254, SEQ ID NO: 256, SEQ ID NO: 258, SEQ ID NO: 260, SEQ ID NO:262, SEQ ID NO: 282, SEQ ID NO: 284 and SEQ ID NO: 286. Most preferredis an allelic variant of a nucleic acid as represented by SEQ ID NO:234.

A further nucleic acid variant useful in the methods of the invention isa nucleic acid variant obtained by gene shuffling. Gene shuffling ordirected evolution may also be used to generate variants of nucleicacids encoding SPL15 transcription factor polypeptides as defined above.

Furthermore, nucleic acid variants may also be obtained by site-directedmutagenesis. Several methods are available to achieve site-directedmutagenesis, the most common being PCR based methods (Current Protocolsin Molecular Biology. Wiley (Eds)).

Also useful in the methods of the invention are nucleic acids encodinghomologues of any one of the amino acids represented by SEQ ID NO: 235,SEQ ID NO: 237, SEQ ID NO: 239, SEQ ID NO: 241, SEQ ID NO: 243, SEQ IDNO: 245, SEQ ID NO: 247, SEQ ID NO: 249, SEQ ID NO: 251, SEQ ID NO: 253,SEQ ID NO: 255, SEQ ID NO: 257, SEQ ID NO: 259, SEQ ID NO: 261, SEQ IDNO: 263, SEQ ID NO: 283, SEQ ID NO: 285, SEQ ID NO: 287, or orthologuesor paralogues of any of the aforementioned SEQ ID NOs.

Also useful in the methods of the invention are nucleic acids encodingderivatives of any one of the amino acids represented by SEQ ID NO: 235,SEQ ID NO: 237, SEQ ID NO: 239, SEQ ID NO: 241, SEQ ID NO: 243, SEQ IDNO: 245, SEQ ID NO: 247, SEQ ID NO: 249, SEQ ID NO: 251, SEQ ID NO: 253,SEQ ID NO: 255, SEQ ID NO: 257, SEQ ID NO: 259, SEQ ID NO: 261 SEQ IDNO: 263, SEQ ID NO: 283, SEQ ID NO: 285, SEQ ID NO: 287, or orthologuesor paralogues of any of the aforementioned SEQ ID NOs. “Derivatives”include peptides, oligopeptides, polypeptides which may, compared to theamino acid sequence of the naturally-occurring form of the protein, suchas the one presented in SEQ ID NO: 235, comprise substitutions of aminoacids with non-naturally occurring amino acid residues, or additions ofnon-naturally occurring amino acid residues.

Furthermore, SPL15 transcription factor polypeptides (at least in theirnative form) typically have DNA-binding activity and an activationdomain. A person skilled in the art may easily determine the presence ofan activation domain and DNA-binding activity using routine tools andtechniques.

Nucleic acids encoding SPL15 transcription factor polypeptides may bederived from any natural or artificial source. The nucleic acid may bemodified from its native form in composition and/or genomic environmentthrough deliberate human manipulation. Preferably the SPL15transcription factor polypeptide-encoding nucleic acid is from a plant,further preferably from a dicot, more preferably from the Brassicaceafamily, most preferably the nucleic acid is from Arabidopsis thaliana.

Any reference herein to a SPL15 transcription factor polypeptide istherefore taken to mean a SPL15 transcription factor peptide as definedabove. Any nucleic acid encoding such a SPL15 transcription factorpolypeptide is suitable for use in performing the methods of theinvention.

The invention also provides genetic constructs and vectors to facilitateintroduction and/or expression of the nucleic acid sequences useful inthe methods according to the invention, in a plant.

Therefore, there is provided a gene construct comprising:

-   -   (i) A nucleic acid encoding a SPL15 transcription factor        polypeptide as defined hereinabove;    -   (ii) One or more control sequences operably liked to the nucleic        acid of (i).

Constructs useful in the methods according to the present invention maybe constructed using recombinant DNA technology well known to personsskilled in the art. The gene constructs may be inserted into vectors,which may be commercially available, suitable for transforming intoplants and suitable for expression of the gene of interest in thetransformed cells. The invention therefore provides use of a geneconstruct as defined hereinabove in the methods of the invention.

Plants are transformed with a vector comprising the sequence of interest(i.e., a nucleic acid encoding a SPL15 transcription factorpolypeptide). The skilled artisan is well aware of the genetic elementsthat must be present on the vector in order to successfully transform,select and propagate host cells containing the sequence of interest. Thesequence of interest is operably linked to one or more control sequences(at least to a promoter).

Advantageously, any type of promoter, whether natural or synthetic, maybe used to drive expression of the nucleic acid sequence. The promotermay be an inducible promoter, i.e. having induced or increasedtranscription initiation in response to a developmental, chemical,environmental or physical stimulus. The promoter may be atissue-specific promoter, i.e. one that is capable of preferentiallyinitiating transcription in certain tissues, such as the leaves, roots,seed tissue etc.

According to the invention, the nucleic acid encoding a SPL15transcription factor polypeptide is operably linked to a constitutivepromoter. The constitutive promoter is preferably a HMGB (high mobilitygroup B) promoter, more preferably the constitutive promoter is a riceHMGB promoter, further preferably the constitutive promoter isrepresented by a nucleic acid sequence substantially similar to SEQ IDNO: 279, most preferably the constitutive promoter is as represented bySEQ ID NO: 279 or SEQ ID NO: 294.

It should be clear that the applicability of the present invention isnot restricted to the nucleic acid encoding an SPL15 transcriptionfactor polypeptide as represented by SEQ ID NO: 234, nor is theapplicability of the invention restricted to expression of a suchnucleic acid encoding an SPL15 transcription factor polypeptide whendriven by a HMGB promoter. Examples of other constitutive promoterswhich may also be used perform the methods of the invention are shown inthe definitions section.

Additional regulatory elements for increasing expression of nucleicacids or genes, or gene products, may include transcriptional as well astranslational enhancers. Those skilled in the art will be aware ofterminator and enhancer sequences that may be suitable for use inperforming the invention. Other control sequences (besides promoter,enhancer, silencer, intron sequences, 3′UTR and/or 5′UTR regions) may beprotein and/or RNA stabilizing elements. Such sequences would be knownor may readily be obtained by a person skilled in the art. Optionally,one or more terminator sequences (also a control sequence) may be usedin the construct introduced into a plant.

An intron sequence may also be added to the 5′ untranslated region (UTR)or the coding sequence of the partial coding sequence to increase theamount of the mature message that accumulates in the cytosol.

The genetic constructs of the invention may further include an origin ofreplication sequence that is required for maintenance and/or replicationin a specific cell type. One example is when a genetic construct isrequired to be maintained in a bacterial cell as an episomal geneticelement (e.g. plasmid or cosmid molecule). Preferred origins ofreplication include, but are not limited to, the f1-ori and colE1.

The genetic construct may optionally comprise a selectable marker gene.

The invention also provides a method for the production of transgenicplants having increased yield relative to control plants, comprisingintroduction and expression in a plant of a nucleic acid encoding aSPL15 transcription factor polypeptide as defined hereinabove.

More specifically, the present invention provides a method for theproduction of transgenic plants having increased yield relative tocontrol plants, which method comprises:

-   -   (i) introducing and expressing a nucleic acid encoding a SPL15        transcription factor polypeptide in a plant cell; and    -   (ii) cultivating the plant cell under conditions promoting plant        growth and development.

The nucleic acid may be introduced directly into a plant cell or intothe plant itself (including introduction into a tissue, organ or anyother part of a plant). According to a preferred feature of the presentinvention, the nucleic acid is preferably introduced into a plant bytransformation. Generally after transformation, plant cells or cellgroupings are selected for the presence of one or more markers which areencoded by plant-expressible genes co-transferred with the gene ofinterest, following which the transformed material is regenerated into awhole plant.

Following DNA transfer and regeneration, putatively transformed plantsmay be evaluated, for instance using Southern analysis, for the presenceof the gene of interest, copy number and/or genomic organisation.Alternatively or additionally, expression levels of the newly introducedDNA may be monitored using Northern and/or Western analysis, orquantitative PCR, all techniques being well known to persons havingordinary skill in the art.

The generated transformed plants may be propagated by a variety ofmeans, such as by clonal propagation or classical breeding techniques.For example, a first generation (or T1) transformed plant may be selfedto give homozygous second generation (or T2) transformants, and the T2plants further propagated through classical breeding techniques.

The generated transformed organisms may take a variety of forms. Forexample, they may be chimeras of transformed cells and non-transformedcells; clonal transformants (e.g., all cells transformed to contain theexpression cassette); grafts of transformed and untransformed tissues(e.g., in plants, a transformed rootstock grafted to an untransformedscion).

The present invention clearly extends to any plant cell or plantproduced by any of the methods described herein, and to all plant partsand propagules thereof. The present invention extends further toencompass the progeny of a primary transformed or transfected cell,tissue, organ or whole plant that has been produced by any of theaforementioned methods, the only requirement being that progeny exhibitthe same genotypic and/or phenotypic characteristic(s) as those producedby the parent in the methods according to the invention.

The invention also includes host cells containing an isolated nucleicacid encoding a SPL15 transcription factor polypeptide as definedhereinabove. Preferred host cells according to the invention are plantcells.

The invention also extends to harvestable parts of a plant such as, butnot limited to seeds, leaves, fruits, flowers, stems, rhizomes, tubersand bulbs. The invention furthermore relates to products derived,preferably directly derived, from a harvestable part of such a plant,such as dry pellets or powders, oil, fat and fatty acids, starch orproteins.

As mentioned above, a preferred method for increasing expression of anucleic acid encoding a SPL15 transcription factor polypeptide is byintroducing and expressing in a plant a nucleic acid encoding a SPL15transcription factor polypeptide; however the effects of performing themethod, i.e. increasing yield, may also be achieved using other wellknown techniques. A description of some of these techniques will nowfollow.

One such technique is T-DNA activation tagging. The resulting transgenicplants show dominant phenotypes due to modified expression of genesclose to the introduced promoter.

The effects of the invention may also be reproduced using the techniqueof TILLING (Targeted Induced Local Lesions In Genomes).

The effects of the invention may also be reproduced using homologousrecombination.

“Increased yield” as defined herein is taken to mean an increase inbiomass (weight) of one or more parts of a plant, which may includeaboveground (harvestable) parts and/or (harvestable) parts below ground.

In particular, such harvestable parts include vegetative biomass and/orseeds, and performance of the methods of the invention results in plantshaving increased yield (in vegetative biomass and/or seed) relative tothe yield of control plants.

Taking corn as an example, a yield increase may be manifested as one ormore of the following: increase in the number of plants per hectare oracre, an increase in the number of ears per plant, an increase in thenumber of rows, number of kernels per row, kernel weight, thousandkernel weight, ear length/diameter, increase in the seed filling rate(which is the number of filled seeds divided by the total number ofseeds and multiplied by 100), among others. Taking rice as an example, ayield increase may manifest itself as an increase in one or more of thefollowing: number of plants per hectare or acre, number of panicles perplant, number of spikelets per panicle, number of flowers (florets) perpanicle (which is expressed as a ratio of the number of filled seedsover the number of primary panicles), increase in the seed filling rate(which is the number of filled seeds divided by the total number ofseeds and multiplied by 100), increase in thousand kernel weight, amongothers.

Since the transgenic plants according to the present invention haveincreased yield, it is likely that these plants exhibit an increasedgrowth rate (during at least part of their life cycle), relative to thegrowth rate of corresponding wild type plants at a corresponding stagein their life cycle. The increased growth rate may be specific to one ormore parts of a plant (including seeds), or may be throughoutsubstantially the whole plant. A plant having an increased growth ratemay even exhibit early flowering. The increase in growth rate may alterthe harvest cycle of a plant allowing plants to be sown later and/orharvested sooner than would otherwise be possible. If the growth rate issufficiently increased, it may allow for the further sowing of seeds ofthe same plant species (for example sowing and harvesting of rice plantsfollowed by sowing and harvesting of further rice plants all within oneconventional growing period). Similarly, if the growth rate issufficiently increased, it may allow for the further sowing of seeds ofdifferent plants species (for example the sowing and harvesting of cornplants followed by, for example, the sowing and optional harvesting ofsoy bean, potato or any other suitable plant). Harvesting additionaltimes from the same rootstock in the case of some crop plants may alsobe possible. Altering the harvest cycle of a plant may lead to anincrease in annual biomass production per acre (due to an increase inthe number of times (say in a year) that any particular plant may begrown and harvested). An increase in growth rate may also allow for thecultivation of transgenic plants in a wider geographical area than theirwild-type counterparts, since the territorial limitations for growing acrop are often determined by adverse environmental conditions either atthe time of planting (early season) or at the time of harvesting (lateseason). Such adverse conditions may be avoided if the harvest cycle isshortened. The growth rate may be determined by deriving variousparameters from growth curves, such parameters may be: T-Mid (the timetaken for plants to reach 50% of their maximal size) and T-90 (timetaken for plants to reach 90% of their maximal size), amongst others.

According to a preferred feature of the present invention, performanceof the methods of the invention gives plants having an increased growthrate relative to control plants. Therefore, according to the presentinvention, there is provided a method for increasing the growth rate ofplants, which method comprises increasing expression in a plant of anucleic acid encoding a SPL15 transcription factor polypeptide

An increase in yield and/or growth rate occurs whether the plant isunder non-stress conditions or whether the plant is exposed to variousstresses compared to control plants. Plants typically respond toexposure to stress by growing more slowly. In conditions of severestress, the plant may even stop growing altogether. Mild stress on theother hand is defined herein as being any stress to which a plant isexposed which does not result in the plant ceasing to grow altogetherwithout the capacity to resume growth. Mild stress in the sense of theinvention leads to a reduction in the growth of the stressed plants ofless than 40%, 35% or 30%, preferably less than 25%, 20% or 15%, morepreferably less than 14%, 13%, 12%, 11% or 10% or less in comparison tothe control plant under non-stress conditions. Due to advances inagricultural practices (irrigation, fertilization, pesticide treatments)severe stresses are not often encountered in cultivated crop plants. Asa consequence, the compromised growth induced by mild stress is often anundesirable feature for agriculture. Mild stresses are the everydaybiotic and/or abiotic (environmental) stresses to which a plant isexposed. Abiotic stresses may be due to drought or excess water,anaerobic stress, salt stress, chemical toxicity, oxidative stress andhot, cold or freezing temperatures. The abiotic stress may be an osmoticstress caused by a water stress (particularly due to drought), saltstress, oxidative stress or an ionic stress. Biotic stresses aretypically those stresses caused by pathogens, such as bacteria, viruses,fungi and insects.

In particular, the methods of the present invention may be performedunder non-stress conditions or under conditions of mild drought to giveplants having increased yield relative to control plants. As reported inWang et al. (Planta (2003) 218: 1-14), abiotic stress leads to a seriesof morphological, physiological, biochemical and molecular changes thatadversely affect plant growth and productivity. Drought, salinity,extreme temperatures and oxidative stress are known to be interconnectedand may induce growth and cellular damage through similar mechanisms.Rabbani et al. (Plant Physiol (2003) 133: 1755-1767) describes aparticularly high degree of “cross talk” between drought stress andhigh-salinity stress. For example, drought and/or salinisation aremanifested primarily as osmotic stress, resulting in the disruption ofhomeostasis and ion distribution in the cell. Oxidative stress, whichfrequently accompanies high or low temperature, salinity or droughtstress, may cause denaturing of functional and structural proteins. As aconsequence, these diverse environmental stresses often activate similarcell signaling pathways and cellular responses, such as the productionof stress proteins, up-regulation of anti-oxidants, accumulation ofcompatible solutes and growth arrest. The term “non-stress” conditionsas used herein are those environmental conditions that allow optimalgrowth of plants. Persons skilled in the art are aware of normal soilconditions and climatic conditions for a given location.

Performance of the methods of the invention gives plants grown undernon-stress conditions or under mild drought conditions increased yieldrelative to control plants grown under comparable conditions. Therefore,according to the present invention, there is provided a method forincreasing yield in plants grown under non-stress conditions or undermild drought conditions, which method comprises increasing expression ina plant of a nucleic acid encoding a MADS15 polypeptide.

Performance of the methods of the invention gives plants grown underconditions of nutrient deficiency, particularly under conditions ofnitrogen deficiency, increased yield relative to control plants grownunder comparable conditions. Therefore, according to the presentinvention, there is provided a method for increasing yield in plantsgrown under conditions of nutrient deficiency, which method comprisesincreasing expression in a plant of a nucleic acid encoding a MADS15polypeptide. Nutrient deficiency may result from a lack or excess ofnutrients such as nitrogen, phosphates and other phosphorous-containingcompounds, potassium, calcium, cadmium, magnesium, manganese, iron andboron, amongst others.

The methods of the invention are advantageously applicable to any plant.

Plants that are particularly useful in the methods of the inventioninclude all plants which belong to the superfamily Viridiplantae, inparticular monocotyledonous and dicotyledonous plants including fodderor forage legumes, ornamental plants, food crops, trees or shrubs.According to a preferred embodiment of the present invention, the plantis a crop plant. Examples of crop plants include soybean, sunflower,canola, alfalfa, rapeseed, cotton, tomato, potato and tobacco. Furtherpreferably, the plant is a monocotyledonous plant. Examples ofmonocotyledonous plants include sugarcane. More preferably the plant isa cereal. Examples of cereals include rice, maize, wheat, barley,millet, rye, triticale, sorghum and oats.

The present invention also encompasses plants obtainable by the methodsaccording to the present invention. The present invention thereforeprovides plants, parts and cells from such plants obtainable by themethods according to the present invention, which plants or parts orcells comprise a nucleic acid transgene encoding a SPL15 transcriptionfactor polypeptide as defined above.

The present invention also encompasses use of nucleic acids encodingSPL15 transcription factor polypeptides in increasing yield in a plantcompared to yield in a control plant.

One such use relates to increasing yield of plants, yield being definedas defined herein above. Yield may in particular include one or more ofthe following: increased aboveground biomass, increased number offlowers per panicle, increased seed yield, increased total number ofseeds, increased number of filled seeds, increased thousand kernelweight (TKW) and increased harvest index.

Nucleic acids encoding SPL15 transcription factor polypeptides may finduse in breeding programmes in which a DNA marker is identified which maybe genetically linked to a gene encoding SPL15 transcription factorpolypeptide. Nucleic acids encoding SPL15 transcription factorpolypeptides may be used to define a molecular marker. This marker maythen be used in breeding programmes to select plants having increasedseed yield. The nucleic acids encoding SPL15 transcription factorpolypeptides may be, for example, a nucleic acid as represented by anyone of SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO: 240,SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO: 246, SEQ ID NO: 248, SEQ IDNO: 250, SEQ ID NO: 252, SEQ ID NO: 254, SEQ ID NO: 256, SEQ ID NO: 258,SEQ ID NO: 260, SEQ ID NO: 262, SEQ ID NO: 282, SEQ ID NO: 284 and SEQID NO: 286.

Allelic variants of a nucleic acid encoding an SPL15 transcriptionfactor polypeptide may also find use in marker-assisted breedingprogrammes. Such breeding programmes sometimes require introduction ofallelic variation by mutagenic treatment of the plants, using forexample EMS mutagenesis; alternatively, the programme may start with acollection of allelic variants of so called “natural” origin causedunintentionally. Identification of allelic variants then takes place,for example, by PCR. This is followed by a step for selection ofsuperior allelic variants of the sequence in question and which giveincreased seed yield. Selection is typically carried out by monitoringgrowth performance of plants containing different allelic variants ofthe sequence in question, for example, different allelic variants of anyone of SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO: 240,SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO: 246, SEQ ID NO: 248, SEQ IDNO: 250, SEQ ID NO: 252, SEQ ID NO: 254, SEQ ID NO: 256, SEQ ID NO: 258,SEQ ID NO: 260, SEQ ID NO: 262, SEQ ID NO: 282, SEQ ID NO: 284 and SEQID NO: 286. Growth performance may be monitored in a greenhouse or inthe field. Further optional steps include crossing plants, in which thesuperior allelic variant was identified, with another plant. This couldbe used, for example, to make a combination of interesting phenotypicfeatures.

Nucleic acids encoding SPL15 transcription factor polypeptides may alsobe used as probes for genetically and physically mapping the genes thatthey are a part of, and as markers for traits linked to those genes.Such information may be useful in plant breeding in order to developlines with desired phenotypes. Such use of nucleic acids encoding SPL15transcription factor polypeptides requires only a nucleic acid sequenceof at least 15 nucleotides in length. The nucleic acids encoding SPL15transcription factor polypeptides may be used as restriction fragmentlength polymorphism (RFLP) markers. Southern blots (Sambrook J, FritschE F and Maniatis T (1989) Molecular Cloning, A Laboratory Manual) ofrestriction-digested plant genomic DNA may be probed with a nucleic acidencoding SPL15 transcription factor polypeptide. The resulting bandingpatterns may then be subjected to genetic analyses using computerprograms such as MapMaker (Lander et al. (1987) Genomics 1: 174-181) inorder to construct a genetic map. In addition, the nucleic acid may beused to probe Southern blots containing restriction endonuclease-treatedgenomic DNAs of a set of individuals representing parent and progeny ofa defined genetic cross. Segregation of the DNA polymorphisms is notedand used to calculate the position of the nucleic acid encoding SPL15transcription factor polypeptide in the genetic map previously obtainedusing this population (Botstein et al. (1980) Am. J. Hum. Genet. 32:314-331).

The production and use of plant gene-derived probes for use in geneticmapping is described in Bernatzky and Tanksley (GENETICS 112 (4):887-898, 1986). Numerous publications describe genetic mapping ofspecific cDNA clones using the methodology outlined above or variationsthereof. For example, F2 intercross populations, backcross populations,randomly mated populations, near isogenic lines (NIL), and other sets ofindividuals may be used for mapping. Such methodologies are well knownto those skilled in the art.

The nucleic acid probes may also be used for physical mapping (i.e.,placement of sequences on physical maps; see Hoheisel et al. In:Non-mammalian Genomic Analysis: A Practical Guide, Academic press 1996,pp. 319-346, and references cited therein).

In another embodiment, the nucleic acid probes may be used in directfluorescence in situ hybridization (FISH) mapping (Trask (1991) TrendsGenet. 7:149-154). Although current methods of FISH mapping favour useof large clones (several kb to several hundred kb; see Laan et al.(1995) Genome Res. 5:13-20), improvements in sensitivity may allowperformance of FISH mapping using shorter probes.

A variety of nucleic acid amplification-based methods for genetic andphysical mapping may be carried out using the nucleic acids. Examplesinclude allele-specific amplification (Kazazian (1989) J. Lab. Clin.Med. 11:95-96), polymorphism of PCR-amplified fragments (CAPS; Sheffieldet al. (1993) Genomics 16:325-332), allele-specific ligation (Landegrenet al. (1988) Science 241:1077-1080), nucleotide extension reactions(Sokolov (1990) Nucleic Acid Res. 18:3671), Radiation Hybrid Mapping(Walter et al. (1997) Nat. Genet. 7:22-28) and Happy Mapping (Dear andCook (1989) Nucleic Acid Res. 17:6795-6807). For these methods, thesequence of a nucleic acid is used to design and produce primer pairsfor use in the amplification reaction or in primer extension reactions.The design of such primers is well known to those skilled in the art. Inmethods employing PCR-based genetic mapping, it may be necessary toidentify DNA sequence differences between the parents of the mappingcross in the region corresponding to the instant nucleic acid sequence.This, however, is generally not necessary for mapping methods.

The methods according to the present invention result in plants havingincreased yield, as described hereinbefore. These traits may also becombined with other economically advantageous traits, such as furtheryield-increasing traits, tolerance to other abiotic and biotic stresses,traits modifying various architectural features and/or biochemicaland/or physiological features.

DESCRIPTION OF FIGURES

The present invention will now be described with reference to thefollowing figures in which:

FIG. 1 represents the schematic structure of the AtHAL3a polypeptidecomprising from N-terminus to C-terminus (i) the substrate binding helix(single underlined), (ii) the insertion His motif (double underlined),(iii) the PXMNXXMW motif (dotted) and (iv) the substrate recognitionclamp (wave underlined). The N-terminal and C-terminal ends of the HAL3proteins are not very conserved.

FIG. 2 shows a binary vector pEXP::HAL3, for increased expression inOryza sativa of an Arabidopsis thaliana HAL3 nucleic acid under thecontrol of a beta expansin promoter.

FIG. 3 shows a multiple alignment of a number of HAL3 sequences fromArabidopsis thaliana AtHAL3b (At_NP_(—)973994), Sorghum bicolor (Sb),Arabidopsis thaliana AtHAL3a (Ath0218), Gossipium hirsutum (Cg), Hordeumvulgare (Hy), Oryza sativa (Os), Vitis vinifera (Vv), Glycine max (Gm),Solanum tuberosum (St), Zea mays (Zm), and Pinus sp (Pg).

FIG. 4 details examples of sequences useful in performing the methodsaccording to the present invention: SEQ ID NO: 1 and SEQ ID NO: 2represent the nucleic acid sequence and the protein sequence of AtHAI3a.SEQ ID NO: 3 and SEQ ID NO: 4 are the sequences of the forward andreverse primers used to isolate the AtHAL3 gene. SEQ ID NO: 5 is thesequence of the beta expansin promoter used in the methods of thepresent invention. SEQ ID NO: 9 to SEQ ID NO: 42 represent examples offull length or partial DNA/protein sequences, useful in the methods ofthe invention or for isolating corresponding full length sequences. Insome cases, sequences were assembled from EST sequences, with lesserquality sequencing. As a consequence, a few nucleic acid substitutionsmay be expected.

FIG. 5 (A) shows the domain structure of the MADS15 protein, (B)represents the sequence of SEQ ID NO: 44 with the MADS domain in boldand the keratin domain in italics. Between the MADS domain and thekeratin, the intervening domain is located while the C-domain is locatedC-terminally of the keratin domain.

FIG. 6 shows a multiple alignment of various MADS15 proteins. Theasterisks indicate identical amino acids, the colons represent highlyconservative substitutions, the dots represent less conservedsubstitutions.

FIG. 7 shows the binary vector for increased expression in Oryza sativaof an Oryza sativa MADS15 protein-encoding nucleic acid under thecontrol of a GOS2 promoter.

FIG. 8 details examples of sequences useful in performing the methodsaccording to the present invention.

FIG. 9 is a schematic representation of a full-length OsMADS15polypeptide. The typical domains (M, MADS; I, intervening domain; K,keratin K-box region; C, variable C-terminal region) are indicated, thelines above and below the diagram show the regions of the proteininvolved in DNA binding, dimerisation and in multimerisation withinteracting proteins.

FIG. 10 shows the binary vector pGOS::MADS15hp for MADS15 RNA silencingin Oryza sativa, using a hairpin construct under the control of aconstitutive promoter (GOS2).

FIG. 11 details examples of sequences useful in performing the methodsaccording to the present invention, or useful in isolating suchsequences. Sequences may result from public EST assemblies, with lesserquality sequencing. As a consequence, a few nucleic acid substitutionsmay be expected. The start (ATG) and stop codons delimit the nucleicacid sequences when these encode full-length MADS15 polypeptides.However both 5′ and 3′ UTR may also be used for the performing themethods of the invention.

FIG. 12 shows the schematic classification of the AP2/ERF transcriptionfactor polypeptides according to the domains present: the AP2 subfamilywith two AP2 repeats and the ERF subfamily with only one AP2 repeat. TheERF subfamily is further subdivided into transcription factorscomprising a B3 domain in addition to the AP2 repeat (called RAV), ornot. The PLT transcription factor polypeptides belong to the AP2subfamily with two AP2 repeats.

FIG. 13 represents a schematic presentation of the PLT transcriptionfactor polypeptide structure. The AP2 domain comprises the two AP2repeats (boxed) separated by a linker region. The approximate positionsof the nuclear localisation signal (NLS), of motif 1 PK(V/L)(A/E)DFLGand of motif 2 (V/L)FX(M/V)WN(D/E) are marked as boxes.

FIG. 14 is an alignment of PLT transcription factor polypeptidesequences (from Table 4), compared to other AP2 domain transcriptionfactor polypeptides (from Table 4 below). The nuclear localisationsignal (NLS), motif 1 PK(V/L)(A/E)DFLG and motif 2 (V/L)FX(M/V)WN(D/E)are boxed. Identical residues are blackened, conservative residues aregrayed.

TABLE 4 AP2 domain transcription factor polypeptides aligned against thePLT transcription factor polypeptides used to perform the methods of theinvention. Name NCBI accession number Source Arath_ANT NM_119937Arabidopsis thaliana Arath_BBM NM_121749.1 Arabidopsis thalianaBrana_BBM1 AF317904 Brassica napus Brana_BBM2 AF317905 Brassica napusMedtr_AP2 BBM AY899909 Medicago truncatula Nicta_ANT like AY461432Nicotiana tabacum Orysa_AP2 XM_473084 Oryza sativa Pinth_ANTL1 AB101585Pinus thunbergii Zeama_AP2 AY109146.1 Zea mays

FIG. 15 is a photograph of 40 seeds from a control plant (left) comparedto 40 seeds from a transgenic plant with increased expression of a PLTtranscription factor polypeptide.

FIG. 16 shows a binary vector pGOS2::PLT, for increased expression inOryza sativa of an Oryza sativa PLT transcription factor nucleic acidunder the control of a GOS2 promoter.

FIG. 17 details examples of sequences useful in performing the methodsaccording to the present invention (SEQ ID NO: 175 to SEQ ID NO: 188).Partial sequences (SEQ ID NO: 189 and SEQ ID NO: 190) useful inisolating corresponding full length sequences are also presented.

FIG. 18 shows an alignment of bHLH transcription factors as definedhereinabove. The sequences were aligned using AlignX program from VectorNTI suite (InforMax, Bethesda, Md.). Multiple alignment was done with agap opening penalty of 10 and a gap extension of 0.01. Minor manualediting was also carried out where necessary to better position someconserved regions. The bHLH domain is indicated within the solid box andthe PFB26111 domain is indicated within the dashed box. Also indicatedby the three upwardly pointing arrows is the 5-9-13 configuration (K/RER). The single downwardly pointing arrow indicates the glutamic acidreside for recognition of the E-BOX.

FIG. 19 shows a matrix of similarity and identity between bHLHtranscription factors from various species. Percentage identity is shownin bold. FIG. 19 a is a matrix over full length sequences and FIG. 19 bis a matrix over the bHLH domain only. More details are provided inExample 34.

FIG. 20 shows a binary vector pGOS2::bHLH, for increased expression inOryza sativa of an Oryza sativa bHLH transcription factor-encodingnucleic acid under the control of a GOS2 promoter.

FIG. 21 details examples of sequences useful in performing the methodsaccording to the present invention.

FIG. 22 Neighbour-joining tree output after a multiple sequencealignment of all Arabidopsis thaliana SPL transcription factorpolypeptides and of SPL15 transcription factor polypeptide orthologuesusing CLUSTAL W (1.83) (at GenomeNet service at the Kyoto UniversityBioinformatics Center), and default values (Blosum 62 as weight matrix,gap open penalty of 10; gap extension penalty of 0.05). Arabidopsisthaliana SPL15 transcription factor polypeptide clusters with the otherSPL15 transcription factor polypeptide orthologues and paralogues, asshown by the curly bracket.

FIG. 23 shows an alignment of the DNA-binding domain (DBD) of SPL15transcription factor polypeptide orthologues and paralogs. The sequenceswere aligned using AlignX program from Vector NTI suite (InforMax,Bethesda, Md.). Multiple alignment was done with a gap opening penaltyof 10 and a gap extension of 0.01. The conserved Cys and His residuesinvolved in zinc ion binding are boxed. The bipartite nuclearlocalization signal (NLS) is underlined.

FIG. 24 is an alignment of SPL15 transcription factor polypeptideorthologues and paralogues. The sequences were aligned using AlignXprogram from Vector NTI suite (InforMax, Bethesda, Md.). Multiplealignment was done with a gap opening penalty of 10 and a gap extensionof 0.01. Minor manual editing was also carried out where necessary tobetter position some conserved regions. The three main characterizeddomains, from N-terminal to C-terminal, are boxed and identified asMotif 1, the SPL DNA binding domain and Motif 2. Additionally, the G/Srich stretch preceding the SPL DBD is marked with Xs and the W(S/T)Ltripeptide at the C-terminal end of the polypeptide also boxed.

FIG. 25 shows a binary vector pHMGB::SPL15, for increased expression inOryza sativa of an Arabidopsis thaliana nucleic acid encoding an SPL15transcription factor polypeptide under the control of an HMGB promoter.

FIG. 26 details examples of sequences useful in performing the methodsaccording to the present invention.

EXAMPLES

The present invention will now be described with reference to thefollowing examples, which are by way of illustration alone. Thefollowing examples are not intended to completely define or otherwiselimit the scope of the invention.

DNA manipulation: unless otherwise stated, recombinant DNA techniquesare performed according to standard protocols described in (Sambrook(2001) Molecular Cloning: a laboratory manual, 3rd Edition Cold SpringHarbor Laboratory Press, CSH, New York) or in Volumes 1 and 2 of Ausubelet al. (1994), Current Protocols in Molecular Biology, CurrentProtocols. Standard materials and methods for plant molecular work aredescribed in Plant Molecular Biology Labfax (1993) by R. D. D. Croy,published by BIOS Scientific Publications Ltd (UK) and BlackwellScientific Publications (UK).

Example Section A HAL3 Example 1 Identification of Sequences Related tothe Nucleic Acid Sequence Used in the Methods of the Invention

Sequences (full length cDNA, ESTs or genomic) related to the nucleicacid sequence used in the methods of the present invention wereidentified amongst those maintained in the Entrez Nucleotides databaseat the National Center for Biotechnology Information (NCBI) usingdatabase sequence search tools, such as the Basic Local Alignment Tool(BLAST) (Altschul et al. (1990) J. Mol. Biol. 215:403-410; and Altschulet al. (1997) Nucleic Acids Res. 25:3389-3402). The program is used tofind regions of local similarity between sequences by comparing nucleicacid or polypeptide sequences to sequence databases and by calculatingthe statistical significance of matches. For example, the polypeptideencoded by the nucleic acid of the present invention was used for theTBLASTN algorithm, with default settings and the filter to ignore lowcomplexity sequences set off. The output of the analysis was viewed bypairwise comparison, and ranked according to the probability score(E-value), where the score reflect the probability that a particularalignment occurs by chance (the lower the E-value, the more significantthe hit). In addition to E-values, comparisons were also scored bypercentage identity. Percentage identity refers to the number ofidentical nucleotides (or amino acids) between the two compared nucleicacid (or polypeptide) sequences over a particular length. In someinstances, the default parameters may be adjusted to modify thestringency of the search. For example the E-value may be increased toshow less stringent matches. This way, short nearly exact matches may beidentified.

Table A provides a list of nucleic acid sequences related to the nucleicacid sequence used in the methods of the present invention.

TABLE A Examples of HAL3 polypeptides: Nucleic acid Plant Source SEQ IDNO: Protein SEQ ID NO: Arabidopsis thaliana 9 10 Oryza sativa 11 12Triticum aestivum 13 14 Zea mays 15 16 Picea abies 17 18 Brassica napus19 20 Brassica oleracea 21 22 Nicotiana tabacum 23 24 Solanum tuberosum25 26 Glycine max 27 28 Vitis vinifera 29 30 Hordeum vulgare 31 32Gossypium hirsutum 33 34 Sorghum bicolor 35 36 Lycopersicon esculentum37 38 Arabidopsis thaliana 39 40 Pinus sp. 41 42

In some instances, related sequences have tentatively been assembled andpublicly disclosed by research institutions, such as The Institute forGenomic Research (TIGR). The Eukaryotic Gene Orthologs (EGO) databasemay be used to identify such related sequences, either by keyword searchor by using the BLAST algorithm with the nucleic acid or polypeptidesequence of interest.

Example 2 Cloning of the Nucleic Acid Sequence Used in the Methods ofthe Invention

The nucleic acid sequence used in the methods of the invention wasamplified by PCR using as template a custom-made Arabidopsis thalianaseedlings cDNA library (in pCMV Sport 6.0; Invitrogen, Paisley, UK). PCRwas performed using Hifi Taq DNA polymerase in standard conditions,using 200 ng of template in a 50 μl PCR mix. The primers used wereprm00957

(SEQ ID NO: 3; sense, start codon in bold: 5′aaaaagcaggctcacaatggagaatgggaaaagagac 3′) andprm00958 (SEQ ID NO: 4; reverse,  complementary,: 5′agaaagctgggttggttttaactagttccaccg 3′),which include the AttB sites for Gateway recombination. The amplifiedPCR fragment was purified also using standard methods. The first step ofthe Gateway procedure, the BP reaction, was then performed, during whichthe PCR fragment recombines in vivo with the pDONR201 plasmid toproduce, according to the Gateway terminology, an “entry clone”, pHAL3.Plasmid pDONR201 was purchased from Invitrogen, as part of the Gateway®technology.

Example 3 Expression Vector Construction

The entry clone pHAL3 was subsequently used in an LR reaction with pEXP,a destination vector used for Oryza sativa transformation. This vectorcontains as functional elements within the T-DNA borders: a plantselectable marker; a screenable marker expression cassette; and aGateway cassette intended for LR in vivo recombination with the nucleicacid sequence of interest already cloned in the entry clone. A rice betaexpansin promoter (SEQ ID NO: 5) for shoot-specific expression waslocated upstream of this Gateway cassette.

After the LR recombination step, the resulting expression vectorpEXP::HAL3 (FIG. 2) was transformed into Agrobacterium strain LBA4044and subsequently to Oryza sativa plants.

Example 4 Crop Transformation

The transformed Agrobacterium containing the expression vectors wereused independently to transform Oryza sativa plants. Mature dry seeds ofthe rice japonica cultivar Nipponbare were dehusked. Sterilization wascarried out by incubating for one minute in 70% ethanol, followed by 30minutes in 0.2% HgCl₂, followed by a 6 times 15 minutes wash withsterile distilled water. The sterile seeds were then germinated on amedium containing 2,4-D (callus induction medium). After incubation inthe dark for four weeks, embryogenic, scutellum-derived calli wereexcised and propagated on the same medium. After two weeks, the calliwere multiplied or propagated by subculture on the same medium foranother 2 weeks. Embryogenic callus pieces were sub-cultured on freshmedium 3 days before co-cultivation (to boost cell division activity).

Agrobacterium strain LBA4404 containing the expression vector was usedfor co-cultivation. Agrobacterium was inoculated on AB medium with theappropriate antibiotics and cultured for 3 days at 28° C. The bacteriawere then collected and suspended in liquid co-cultivation medium to adensity (OD₆₀₀) of about 1. The suspension was then transferred to aPetri dish and the calli immersed in the suspension for 15 minutes. Thecallus tissues were then blotted dry on a filter paper and transferredto solidified, co-cultivation medium and incubated for 3 days in thedark at 25° C. Co-cultivated calli were grown on 2,4-D-containing mediumfor 4 weeks in the dark at 28° C. in the presence of a selection agent.During this period, rapidly growing resistant callus islands developed.After transfer of this material to a regeneration medium and incubationin the light, the embryogenic potential was released and shootsdeveloped in the next four to five weeks. Shoots were excised from thecalli and incubated for 2 to 3 weeks on an auxin-containing medium fromwhich they were transferred to soil. Hardened shoots were grown underhigh humidity and short days in a greenhouse.

The primary transformants were transferred from a tissue culture chamberto a greenhouse. After a quantitative PCR analysis to verify copy numberof the T-DNA insert, only single copy transgenic plants that exhibittolerance to the selection agent were kept for harvest of T1 seed. Seedswere then harvested three to five months after transplanting. The methodyielded single locus transformants at a rate of over 50% (Aldemita andHodges 1996, Chan et al. 1993, Hiei et al. 1994).

Corn Transformation

Transformation of maize (Zea mays) is performed with a modification ofthe method described by Ishida et al. (1996) Nature Biotech 14(6):745-50. Transformation is genotype-dependent in corn and only specificgenotypes are amenable to transformation and regeneration. The inbredline A188 (University of Minnesota) or hybrids with A188 as a parent aregood sources of donor material for transformation, but other genotypescan be used successfully as well. Ears are harvested from corn plantapproximately 11 days after pollination (DAP) when the length of theimmature embryo is about 1 to 1.2 mm. Immature embryos are cocultivatedwith Agrobacterium tumefaciens containing the expression vector, andtransgenic plants are recovered through organogenesis. Excised embryosare grown on callus induction medium, then maize regeneration medium,containing the selection agent (for example imidazolinone but variousselection markers can be used). The Petri plates are incubated in thelight at 25° C. for 2-3 weeks, or until shoots develop. The green shootsare transferred from each embryo to maize rooting medium and incubatedat 25° C. for 2-3 weeks, until roots develop. The rooted shoots aretransplanted to soil in the greenhouse. T1 seeds are produced fromplants that exhibit tolerance to the selection agent and that contain asingle copy of the T-DNA insert.

Wheat Transformation

Transformation of wheat is performed with the method described by Ishidaet al. (1996) Nature Biotech 14(6): 745-50. The cultivar Bobwhite(available from CIMMYT, Mexico) is commonly used in transformation.Immature embryos are co-cultivated with Agrobacterium tumefacienscontaining the expression vector, and transgenic plants are recoveredthrough organogenesis. After incubation with Agrobacterium, the embryosare grown in vitro on callus induction medium, then regeneration medium,containing the selection agent (for example imidazolinone but variousselection markers can be used). The Petri plates are incubated in thelight at 25° C. for 2-3 weeks, or until shoots develop. The green shootsare transferred from each embryo to rooting medium and incubated at 25°C. for 2-3 weeks, until roots develop. The rooted shoots aretransplanted to soil in the greenhouse. T1 seeds are produced fromplants that exhibit tolerance to the selection agent and that contain asingle copy of the T-DNA insert.

Soybean Transformation

Soybean is transformed according to a modification of the methoddescribed in the Texas A&M U.S. Pat. No. 5,164,310. Several commercialsoybean varieties are amenable to transformation by this method. Thecultivar Jack (available from the Illinois Seed foundation) is commonlyused for transformation. Soybean seeds are sterilised for in vitrosowing. The hypocotyl, the radicle and one cotyledon are excised fromseven-day old young seedlings. The epicotyl and the remaining cotyledonare further grown to develop axillary nodes. These axillary nodes areexcised and incubated with Agrobacterium tumefaciens containing theexpression vector. After the cocultivation treatment, the explants arewashed and transferred to selection media. Regenerated shoots areexcised and placed on a shoot elongation medium. Shoots no longer than 1cm are placed on rooting medium until roots develop. The rooted shootsare transplanted to soil in the greenhouse. T1 seeds are produced fromplants that exhibit tolerance to the selection agent and that contain asingle copy of the T-DNA insert.

Rapeseed/Canola Transformation

Cotyledonary petioles and hypocotyls of 5-6 day old young seedling areused as explants for tissue culture and transformed according to Babicet al. (1998, Plant Cell Rep 17: 183-188). The commercial cultivarWestar (Agriculture Canada) is the standard variety used fortransformation, but other varieties can also be used. Canola seeds aresurface-sterilized for in vitro sowing. The cotyledon petiole explantswith the cotyledon attached are excised from the in vitro seedlings, andinoculated with Agrobacterium (containing the expression vector) bydipping the cut end of the petiole explant into the bacterialsuspension. The explants are then cultured for 2 days on MSBAP-3 mediumcontaining 3 mg/l BAP, 3% sucrose, 0.7% Phytagar at 23° C., 16 hr light.After two days of co-cultivation with Agrobacterium, the petioleexplants are transferred to MSBAP-3 medium containing 3 mg/l BAP,cefotaxime, carbenicillin, or timentin (300 mg/l) for 7 days, and thencultured on MSBAP-3 medium with cefotaxime, carbenicillin, or timentinand selection agent until shoot regeneration. When the shoots are 5-10mm in length, they are cut and transferred to shoot elongation medium(MSBAP-0.5, containing 0.5 mg/l BAP). Shoots of about 2 cm in length aretransferred to the rooting medium (MS0) for root induction. The rootedshoots are transplanted to soil in the greenhouse. T1 seeds are producedfrom plants that exhibit tolerance to the selection agent and thatcontain a single copy of the T-DNA insert.

Alfalfa Transformation

A regenerating clone of alfalfa (Medicago sativa) is transformed usingthe method of (McKersie et al., 1999 Plant Physiol 119: 839-847).Regeneration and transformation of alfalfa is genotype dependent andtherefore a regenerating plant is required. Methods to obtainregenerating plants have been described. For example, these can beselected from the cultivar Rangelander (Agriculture Canada) or any othercommercial alfalfa variety as described by Brown D C W and A Atanassov(1985. Plant Cell Tissue Organ Culture 4: 111-112). Alternatively, theRA3 variety (University of Wisconsin) has been selected for use intissue culture (Walker et al., 1978 Am J Bot 65:654-659). Petioleexplants are cocultivated with an overnight culture of Agrobacteriumtumefaciens C58C1 pMP90 (McKersie et al., 1999 Plant Physiol 119:839-847) or LBA4404 containing the expression vector. The explants arecocultivated for 3 d in the dark on SH induction medium containing 288mg/L Pro, 53 mg/L thioproline, 4.35 g/L K₂SO₄, and 100 μmacetosyringinone. The explants are washed in half-strengthMurashige-Skoog medium (Murashige and Skoog, 1962) and plated on thesame SH induction medium without acetosyringinone but with a suitableselection agent and suitable antibiotic to inhibit Agrobacterium growth.After several weeks, somatic embryos are transferred to BOi2Ydevelopment medium containing no growth regulators, no antibiotics, and50 g/L sucrose. Somatic embryos are subsequently germinated onhalf-strength Murashige-Skoog medium. Rooted seedlings were transplantedinto pots and grown in a greenhouse. T1 seeds are produced from plantsthat exhibit tolerance to the selection agent and that contain a singlecopy of the T-DNA insert.

Example 5 Evaluation Procedure 5.1 Evaluation Setup

Approximately 30 independent T0 rice transformants were generated. Theprimary transformants were transferred from a tissue culture chamber toa greenhouse for growing and harvest of T1 seed. Seven events, of whichthe T1 progeny segregated 3:1 for presence/absence of the transgene,were retained. For each of these events, approximately 10 T1 seedlingscontaining the transgene (hetero- and homo-zygotes) and approximately 10T1 seedlings lacking the transgene (nullizygotes) were selected bymonitoring visual marker expression. The transgenic plants and thecorresponding nullizygotes were grown side-by-side at random positions.Greenhouse conditions were of shorts days (12 hours light), 28° C. inthe light and 22° C. in the dark, and a relative humidity of 70%.

Four T1 events were further evaluated in the T2 generation following thesame evaluation procedure as for the T1 generation but with moreindividuals per event. From the stage of sowing until the stage ofmaturity the plants were passed several times through a digital imagingcabinet. At each time point digital images (2048×1536 pixels, 16 millioncolours) were taken of each plant from at least 6 different angles.

5.2 Statistical Analysis: t Test and F Test

A two factor ANOVA (analysis of variants) was used as a statisticalmodel for the overall evaluation of plant phenotypic characteristics. AnF test was carried out on all the parameters measured of all the plantsof all the events transformed with the gene of the present invention.The F test was carried out to check for an effect of the gene over allthe transformation events and to verify for an overall effect of thegene, also known as a global gene effect. The threshold for significancefor a true global gene effect was set at a 5% probability level for theF test. A significant F test value points to a gene effect, meaning thatit is not only the mere presence or position of the gene that is causingthe differences in phenotype.

To check for an effect of the genes within an event, i.e., for aline-specific effect, a t-test was performed within each event usingdata sets from the transgenic plants and the corresponding null plants.“Null plants” or “null segregants” or “nullizygotes” are the plantstreated in the same way as the transgenic plant, but from which thetransgene has segregated. Null plants can also be described as thehomozygous negative transformed plants. The threshold for significancefor the t-test is set at 10% probability level. The results for someevents can be above or below this threshold. This is based on thehypothesis that a gene might only have an effect in certain positions inthe genome, and that the occurrence of this position-dependent effect isnot uncommon. This kind of gene effect is also named herein a “lineeffect of the gene”. The p-value is obtained by comparing the t-value tothe t-distribution or alternatively, by comparing the F-value to theF-distribution. The p-value then gives the probability that the nullhypothesis (i.e., that there is no effect of the transgene) is correct.

Example 6 Evaluation Results

The mature primary panicles were harvested, counted, bagged,barcode-labeled and then dried for three days in an oven at 37° C. Thepanicles were then threshed and all the seeds were collected andcounted. The filled husks were separated from the empty ones using anair-blowing device. The empty husks were discarded and the remainingfraction was counted again. The filled husks were weighed on ananalytical balance. The number of filled seeds was determined bycounting the number of filled husks that remained after the separationstep. The total seed yield (total seed weight) was measured by weighingall filled husks harvested from a plant. Total seed number per plant wasmeasured by counting the number of husks harvested from a plant. Theharvest index (HI) in the present invention is defined as the ratiobetween the total seed yield and the above ground area (mm²), multipliedby a factor 10⁶. The total number of flowers per panicle as defined inthe present invention is the ratio between the total number of seeds andthe number of mature primary panicles. The seed fill rate as defined inthe present invention is the proportion (expressed as a %) of the numberof filled seeds over the total number of seeds (or florets).

As presented in Table B, the seed yield, number of filled seeds andharvest index are increased in the transgenic plants preferentiallyexpressing a nucleic acid encoding a HAL3 polypeptide in the shoot,compared to control plants.

Table B shows the average yield increase (total seed weight), increasein number of filled seeds and increase of harvest index in percent,calculated from the transgenic events compared to control plants, in theT1 generation

TABLE B parameters % increase p-value Total seed weight 14 0.0072 Numberof filled seeds 15 0.0052 Harvest Index 10 0.0093

Example 7 Early Vigour Evaluation Results

Early vigour was determined by counting the total number of pixels fromaboveground plant parts discriminated from the background. This valuewas averaged for the pictures taken on the same time point fromdifferent angles and was converted to a physical surface value expressedin square mm by calibration. The results described below are for plantsthree weeks post-germination.

Plant early vigour (as determined by aboveground area) was seen in sixout of seven events of the T1 generation, with an overall increase inaboveground area for transgenic seedlings of 27% compared to controlplants. Four of these T1 events were further evaluated in the T2generation, and all four of these events gave an increase in abovegroundarea for transgenic seedlings compared to control plants, with anoverall increase in aboveground area for transgenic seedlings of 33%compared to control plants. The results were also shown to bestatistically significant with the p-value from the F-test being lowerthan 0.0001 (T2 generation) indicating that the effect seen is likelydue to the transgene rather than the position of the gene or a lineeffect, see table C.

TABLE C parameters % increase p-value Early vigour 33 0.0000

Example Section B MADS15 Upregulated Example 8 Identification ofSequences Related to SEQ ID NO: 43 and SEQ ID NO: 44

Sequences (full length cDNA, ESTs or genomic) related to SEQ ID NO: 43and/or protein sequences related to SEQ ID NO: 44 were identifiedamongst those maintained in the Entrez Nucleotides database at theNational Center for Biotechnology Information (NCBI) using databasesequence search tools, such as the Basic Local Alignment Tool (BLAST)(Altschul et al. (1990) J. Mol. Biol. 215:403-410; and Altschul et al.(1997) Nucleic Acids Res. 25:3389-3402). The program is used to findregions of local similarity between sequences by comparing nucleic acidor polypeptide sequences to sequence databases and by calculating thestatistical significance of matches. The polypeptide encoded by SEQ IDNO: 43 was used for the TBLASTN algorithm, with default settings and thefilter to ignore low complexity sequences set off. The output of theanalysis was viewed by pairwise comparison, and ranked according to theprobability score (E-value), where the score reflects the probabilitythat a particular alignment occurs by chance (the lower the E-value, themore significant the hit). In addition to E-values, comparisons werealso scored by percentage identity. Percentage identity refers to thenumber of identical nucleotides (or amino acids) between the twocompared nucleic acid (or polypeptide) sequences over a particularlength. In some instances, the default parameters may be adjusted tomodify the stringency of the search.

In addition to the publicly available nucleic acid sequences availableat NCBI, proprietary sequence databases are also searched following thesame procedure as described herein above.

Table D provides a list of nucleic acid and protein sequences related tothe nucleic acid sequence as represented by SEQ ID NO: 43 and theprotein sequence represented by SEQ ID NO: 44.

TABLE D Nucleic acid sequences related to the nucleic acid sequence (SEQID NO: 43) useful in the methods of the present invention, and thecorresponding deduced polypeptides. database SEQ ID NO: accession namesource organism nucl/protein number VRN-H1 Hordeum vulgare 52/53AAW82995 m5 Hordeum vulgare 54/55 CAB97352 TaMADS#11 Triticum aestivum56/57 BAA33457 TaVRT-1 Triticum aestivum 58/59 AAP33790 AP1 Triticummonococcum 60/61 AAO72630 VRN-A1 Triticum aestivum 62/63 AAW73222 MADS1Lolium perenne 64/65 AAO45873 MADS1 Lolium temulentum 66/67 AAD10625 m15Zea mays 68/69 CAD23408 m4 Zea mays 70/71 CAD23417 RMADS211 Oryza sativa72/73 AAS59822 MADS14 Oryza sativa 74/75 AAF19047 Mads2 Dendrocalamuslatiflorus 76/77 AAR32119 Mads1 Dendrocalamus latiflorus 78/79 AAR32118mads3 Zea mays 80/81 AAG43200 SbMADS2 Sorghum bicolor 82/83 AAB50181ZAP1 Zea mays 84/85 AAB00081 MADS2 Lolium temulentum 86/87 AAD10626MADS2 Lolium perenne 88/89 AAO45874 m8 Hordeum vulgare 90/91 CAB97354FDRMADS3 Oryza sativa 92/93 AAL09473 MADS15 Oryza sativa 94/95 AAL09473TvFL2 Tradescantia virginiana 96/97 AAP83415 TvFL1 Tradescantiavirginiana 98/99 AAP83414 TvFL3 Tradescantia virginiana 100/101 AAP83416SQUA1 Elaeis guineensis 102/103 AAQ03221 AIFL Allium sp. 104/105AAP83362 DOMADS2 Dendrobium grex 106/107 AAF13261

Example 9 Alignment of Relevant Polypeptide Sequences

AlignX from the Vector NTI (Invitrogen) is based on the popular Clustalalgorithm of progressive alignment (Thompson et al. (1997) Nucleic AcidsRes 25:4876-4882; Chenna et al. (2003). Nucleic Acids Res 31:3497-3500).A phylogenetic tree can be constructed using a neighbour-joiningclustering algorithm. Default values are for the gap open penalty of 10,for the gap extension penalty of 0.1 and the selected weight matrix isBlosum 62 (if polypeptides are aligned).

The result of the multiple sequence alignment using polypeptidesrelevant in identifying the ones useful in performing the methods of theinvention is shown in FIG. 6.

Example 10 Calculation of Global Percentage Identity Between PolypeptideSequences Useful in Performing the Methods of the Invention

Global percentages of similarity and identity between full lengthpolypeptide sequences useful in performing the methods of the inventionwere determined using one of the methods available in the art, theMatGAT (Matrix Global Alignment Tool) software (BMC Bioinformatics. 20034:29. MatGAT: an application that generates similarity/identity matricesusing protein or DNA sequences. Campanella J J, Bitincka L, Smalley J;software hosted by Ledion Bitincka). MatGAT software generatessimilarity/identity matrices for DNA or protein sequences withoutneeding pre-alignment of the data. The program performs a series ofpair-wise alignments using the Myers and Miller global alignmentalgorithm (with a gap opening penalty of 12, and a gap extension penaltyof 2), calculates similarity and identity using for example Blosum 62(for polypeptides), and then places the results in a distance matrix.Sequence similarity is shown in the bottom half of the dividing line andsequence identity is shown in the top half of the diagonal dividingline.

Parameters used in the comparison were:

-   -   Scoring matrix: Blosum62    -   First Gap: 12    -   Extending gap: 2

Results of the software analysis are shown in Table E for the globalsimilarity and identity over the full length of the polypeptidesequences (excluding the partial polypeptide sequences). Percentageidentity is given above the diagonal and percentage similarity is givenbelow the diagonal.

The percentage identity between the polypeptide sequences useful inperforming the methods of the invention can be as low as 40% amino acididentity compared to SEQ ID NO: 44.

TABLE E MatGAT results for global similarity and identity over the fulllength of the polypeptide sequences. 1 2 3 4 5 6 7 8 9 10 11 12 13 1.SEQID44 70.8 70.0 70.4 70.4 71.5 71.5 70.1 71.6 71.9 72.3 71.5 73.0 2.SEQID53 77.2 99.2 96.3 96.3 95.5 95.1 87.0 86.2 80.6 80.2 81.0 82.9 3.SEQID55 76.4 99.2 95.5 95.5 94.7 94.3 86.2 85.4 79.8 79.4 80.2 82.1 4.SEQID57 76.0 96.7 95.9 98.8 98.0 97.5 87.0 86.6 81.8 81.4 80.6 82.5 5.SEQID59 76.4 97.1 96.3 99.2 98.0 98.0 87.0 86.6 81.8 81.4 80.6 82.5 6.SEQID61 77.2 96.7 95.9 98.4 98.8 99.6 87.9 87.4 82.6 82.6 81.0 82.9 7.SEQID63 77.2 96.3 95.5 98.0 98.8 99.6 87.4 87.0 82.2 82.2 80.6 82.5 8.SEQID65 76.4 92.2 91.4 92.2 92.7 93.5 93.1 97.6 81.7 80.9 82.3 83.8 9.SEQID67 76.8 92.7 91.8 92.2 92.7 93.5 93.1 98.8 81.3 81.7 82.3 83.8 10.SEQID69 79.4 87.3 86.5 89.0 89.0 89.4 89.0 88.2 88.2 93.5 83.8 85.8 11.SEQID71 79.4 87.3 86.5 89.0 89.0 89.4 89.0 86.9 87.3 97.1 83.8 85.8 12.SEQID73 80.1 88.1 87.4 86.6 87.0 87.4 87.0 88.5 88.1 91.3 90.9 96.4 13.SEQID75 79.8 89.8 89.0 88.6 89.0 89.4 89.0 90.7 90.2 93.5 93.1 96.4 14.SEQID77 79.8 91.8 91.0 91.4 91.8 92.6 92.2 90.6 90.2 91.8 91.4 93.3 95.115. SEQID79 80.1 92.2 91.4 91.8 92.2 93.0 92.6 91.0 90.6 92.2 91.8 93.795.5 16. SEQID81 86.3 79.3 78.5 77.8 78.1 78.9 78.9 77.0 77.0 81.9 80.780.7 80.7 17. SEQID85 85.7 76.9 76.2 76.6 76.9 77.7 77.7 75.5 75.1 80.279.1 79.1 79.1 18. SEQID87 89.1 78.2 77.4 79.3 79.7 79.7 80.1 78.2 77.882.8 80.8 81.6 80.8 19. SEQID89 89.5 78.2 77.4 78.9 78.9 79.3 79.3 78.578.2 83.1 81.2 82.0 81.2 20. SEQID91 85.9 75.4 74.6 75.7 75.7 76.4 76.475.4 74.6 79.3 78.6 79.0 79.0 21. SEQID93 91.8 73.0 72.3 73.8 73.8 74.574.5 73.0 73.0 76.0 76.8 78.3 76.4 22. SEQID95 99.6 76.8 76.0 76.0 76.076.8 76.8 76.0 75.7 78.7 79.0 79.8 79.4 23. SEQID103 75.3 82.8 82.0 81.682.0 82.4 82.4 84.4 84.4 82.4 82.4 85.4 84.0 24. SEQID107 70.4 77.3 76.576.5 74.5 77.3 76.1 74.9 74.9 76.5 75.7 75.1 77.3 14 15 16 17 18 19 2021 22 23 24 1. SEQID44 72.7 73.0 82.1 82.2 87.4 87.7 82.5 90.3 99.6 64.756.7 2. SEQID53 84.6 85.0 70.7 68.9 71.3 71.3 68.8 65.2 70.4 70.1 61.03. SEQID55 83.7 84.1 70.0 68.1 70.5 70.5 68.1 64.4 69.7 69.3 60.2 4.SEQID57 85.8 86.2 70.4 69.6 72.4 72.4 69.6 65.5 70.0 69.7 62.0 5.SEQID59 85.8 86.2 70.7 69.6 72.4 72.4 69.6 65.5 70.0 70.1 61.5 6.SEQID61 87.0 87.4 71.1 70.0 72.4 72.4 70.3 67.2 71.2 70.9 62.5 7.SEQID63 86.6 87.0 71.1 70.0 72.8 72.4 70.3 67.2 71.2 70.9 61.8 8.SEQID65 85.7 86.1 70.5 69.3 71.4 71.8 69.3 66.8 69.8 71.8 60.7 9.SEQID67 85.7 86.1 71.2 70.1 72.1 72.5 68.6 66.0 70.5 71.8 60.7 10.SEQID69 84.9 85.3 71.5 71.4 73.9 74.3 69.9 67.8 71.9 71.8 62.5 11.SEQID71 84.5 84.9 71.5 71.4 73.6 73.9 69.6 69.3 71.9 72.2 61.0 12.SEQID73 89.3 89.7 72.0 71.1 72.4 72.8 69.9 68.0 71.2 71.9 60.2 13.SEQID75 91.5 91.9 73.0 71.8 73.9 74.3 70.7 68.3 72.7 72.0 60.6 14.SEQID77 99.6 71.9 71.1 73.6 73.6 69.9 67.8 72.7 73.3 61.8 15. SEQID7999.6 72.2 71.4 73.9 73.9 70.3 68.2 72.7 73.7 62.2 16. SEQID81 79.3 79.693.4 81.9 82.3 79.0 76.8 81.8 64.3 56.8 17. SEQID85 77.7 78.0 96.3 82.582.9 81.3 77.0 81.9 63.6 57.2 18. SEQID87 80.5 80.8 87.4 86.1 99.6 88.482.5 87.0 64.6 58.0 19. SEQID89 80.5 80.8 87.8 86.4 99.6 88.8 82.9 87.464.6 58.0 20. SEQID91 76.8 77.2 86.2 88.0 90.2 90.6 77.5 82.1 62.7 55.821. SEQID93 76.0 76.4 83.3 82.1 85.4 85.8 81.5 89.9 61.0 54.1 22.SEQID95 79.8 79.8 85.9 85.3 88.8 89.1 85.5 91.4 64.3 56.3 23. SEQID10384.8 85.2 75.2 73.6 75.9 75.9 73.6 72.3 74.9 70.1 24. SEQID107 78.5 78.971.5 71.4 72.8 72.8 70.3 67.4 70.0 82.4

Example 11 Identification of Domains Comprised in Polypeptide SequencesUseful in Performing the Methods of the Invention

The Integrated Resource of Protein Families, Domains and Sites(InterPro) database is an integrated interface for the commonly usedsignature databases for text- and sequence-based searches. The InterProdatabase combines these databases, which use different methodologies andvarying degrees of biological information about well-characterizedproteins to derive protein signatures. Collaborating databases includeSWISS-PROT, PROSITE, TrEMBL, PRINTS, Propom and Pfam, Smart andTIGRFAMs. Interpro is hosted at the European Bioinformatics Institute inthe United Kingdom.

The results of the InterPro scan of the polypeptide sequence asrepresented by SEQ ID NO: 44 are presented in Table F.

TABLE F InterPro scan results of the polypeptide sequence as representedby SEQ ID NO: 44 Database Accession number Accession name PRINTS PR00404MADSDOMAIN PFAM PF00319 SRF-TF SMART SM00432 MADS PROFILE PS50066MADS_BOX_2 SUPERFAMILY SSF55455 SRF-like PFAM PF01486 K-box SUPERFAMILYSSF46589 tRNA-binding arm PANTHER PTHR11945 MADS BOX PROTEIN PANTHERPTHR11945:SF19 MADS BOX PROTEIN

Example 12 Topology Prediction of the Polypeptide Sequences Useful inPerforming the Methods of the Invention (Subcellular Localization,Transmembrane . . . )

TargetP 1.1 predicts the subcellular location of eukaryotic proteins.The location assignment is based on the predicted presence of any of theN-terminal pre-sequences: chloroplast transit peptide (cTP),mitochondrial targeting peptide (mTP) or secretory pathway signalpeptide (SP). Scores on which the final prediction is based are notreally probabilities, and they do not necessarily add to one. However,the location with the highest score is the most likely according toTargetP, and the relationship between the scores (the reliability class)may be an indication of how certain the prediction is. The reliabilityclass (RC) ranges from 1 to 5, where 1 indicates the strongestprediction. TargetP is maintained at the server of the TechnicalUniversity of Denmark.

For the sequences predicted to contain an N-terminal presequence apotential cleavage site can also be predicted.

A number of parameters were selected, such as organism group (non-plantor plant), cutoff sets (none, predefined set of cutoffs, oruser-specified set of cutoffs), and the calculation of prediction ofcleavage sites (yes or no).

The results of TargetP 1.1 analysis of the polypeptide sequence asrepresented by SEQ ID NO: 44 are presented Table G. The “plant” organismgroup has been selected, no cutoffs defined, and the predicted length ofthe transit peptide requested. The subcellular localization of thepolypeptide sequence as represented by SEQ ID NO: 44 may be thecytoplasm or nucleus, no transit peptide is predicted.

TABLE G TargetP 1.1 analysis of the polypeptide sequence as representedby SEQ ID NO: 44 Length (AA) 267 Chloroplastic transit peptide 0.088Mitochondrial transit peptide 0.492 Secretory pathway signal peptide0.046 Other subcellular targeting 0.655 Predicted Location ChloroplasticReliability class 5 Predicted transit peptide length /

Many other algorithms can be used to perform such analyses, including:

-   -   ChloroP 1.1 hosted on the server of the Technical University of        Denmark;    -   Protein Prowler Subcellular Localisation Predictor version 1.2        hosted on the server of the Institute for Molecular Bioscience,        University of Queensland, Brisbane, Australia;    -   PENCE Proteome Analyst PA-GOSUB 2.5 hosted on the server of the        University of Alberta, Edmonton, Alberta, Canada;    -   TMHMM, hosted on the server of the Technical University of        Denmark

Example 13 Assay Related to the Polypeptide Sequences Useful inPerforming the Methods of the Invention

Lim et al. (PMB 44, 513-527, 2000) describe two assays for measuringprotein-protein interactions that may be applied to MADS15. In the yeasttwo-hybrid assay, a truncated form of MADS1 (comprising the completeMADS box, the I- and K-domain but lacking the C-terminal part of theC-domain) was used as bait to test interaction with MADS15. In the invitro pull-down assay, GST and GST-fused MADS1 proteins were producedand immobilised on glutathione Sepharose 4B. The resin-boundGST/GST-MADS1 was mixed with ³⁵S-labeled MADS15 proteins or fragmentsthereof. After washing, the interacting proteins were eluted andanalysed by SDS-PAGE. A person skilled in the art will appreciate thatthe MADS15 protein may be used as bait in the two-hybrid screen or maybe immobilised on glutathione resin as well. Furthermore, a MADS15protein, when used according to the methods of the present invention,will result in increased root yield in rice, measured as an increasedratio of root biomass over shoot biomass.

Example 14 Cloning of Nucleic Acid Sequence as Represented by SEQ ID NO:43

The Oryza sativa MADS15 gene was amplified by PCR using as template anOryza sativa seedling cDNA library (Invitrogen, Paisley, UK). Afterreverse transcription of RNA extracted from seedlings, the cDNAs werecloned into pCMV Sport 6.0. Average insert size of the bank was 1.5 kband the original number of clones was of the order of 1.59×10⁷ cfu.Original titer was determined to be 9.6×10⁵ cfu/ml after firstamplification of 6×10¹¹ cfu/ml. After plasmid extraction, 200 ng oftemplate was used in a 50 μl PCR mix. Primers prm06892 (SEQ ID NO: 45;sense, start codon in bold, AttB1 site in italic:5′-ggggacaagtttgtacaaaaaagcaggcttaaaca atgggcgggggaaggt-3′) and prm06893(SEQ ID NO: 46; reverse, complementary, AttB2 site in italic:5′-ggggaccactttgtacaagaaagctgggtttggccgacgacgacgac-3′), which includethe AttB sites for Gateway recombination, were used for PCRamplification. PCR was performed using Hifi Taq DNA polymerase instandard conditions. A PCR fragment of around 915 bp was amplified andpurified also using standard methods. The first step of the Gatewayprocedure, the BP reaction, was then performed, during which the PCRfragment recombines in vivo with the pDONR201 plasmid to produce,according to the Gateway terminology, an “entry clone”, pMADS15. PlasmidpDONR201 was purchased from Invitrogen, as part of the Gateway®technology.

Example 15 Expression Vector Construction Using the Nucleic AcidSequence as Represented by SEQ ID NO: 43

The entry clone pMADS15 was subsequently used in an LR reaction withpGOS2, a destination vector used for Oryza sativa transformation. Thisvector contains as functional elements within the T-DNA borders: a plantselectable marker; a screenable marker expression cassette; and aGateway cassette intended for LR in vivo recombination with the nucleicacid sequence of interest already cloned in the entry clone. A rice GOS2promoter (SEQ ID NO: 108, alternatively, SEQ ID NO: 47 is equallyuseful) for constitutive expression was located upstream of this Gatewaycassette.

After the LR recombination step, the resulting expression vectorpGOS2::MADS15 (FIG. 7) was transformed into Agrobacterium strain LBA4044according to methods well known in the art.

Example 16 Plant Transformation Rice Transformation

The Agrobacterium containing the expression vector was used to transformOryza sativa plants. Mature dry seeds of the rice japonica cultivarNipponbare were dehusked. Sterilization was carried out by incubatingfor one minute in 70% ethanol, followed by 30 minutes in 0.2% HgCl₂,followed by a 6 times 15 minutes wash with sterile distilled water. Thesterile seeds were then germinated on a medium containing 2,4-D (callusinduction medium). After incubation in the dark for four weeks,embryogenic, scutellum-derived calli were excised and propagated on thesame medium. After two weeks, the calli were multiplied or propagated bysubculture on the same medium for another 2 weeks. Embryogenic calluspieces were sub-cultured on fresh medium 3 days before co-cultivation(to boost cell division activity).

Agrobacterium strain LBA4404 containing the expression vector was usedfor co-cultivation. Agrobacterium was inoculated on AB medium with theappropriate antibiotics and cultured for 3 days at 28° C. The bacteriawere then collected and suspended in liquid co-cultivation medium to adensity (OD₆₀₀) of about 1. The suspension was then transferred to aPetri dish and the calli immersed in the suspension for 15 minutes. Thecallus tissues were then blotted dry on a filter paper and transferredto solidified, co-cultivation medium and incubated for 3 days in thedark at 25° C. Co-cultivated calli were grown on 2,4-D-containing mediumfor 4 weeks in the dark at 28° C. in the presence of a selection agent.During this period, rapidly growing resistant callus islands developed.After transfer of this material to a regeneration medium and incubationin the light, the embryogenic potential was released and shootsdeveloped in the next four to five weeks. Shoots were excised from thecalli and incubated for 2 to 3 weeks on an auxin-containing medium fromwhich they were transferred to soil. Hardened shoots were grown underhigh humidity and short days in a greenhouse.

The primary transformants were transferred from a tissue culture chamberto a greenhouse. After a quantitative PCR analysis to verify copy numberof the T-DNA insert, only single copy transgenic plants that exhibittolerance to the selection agent were kept for harvest of T1 seed. Seedswere then harvested three to five months after transplanting. The methodyielded single locus transformants at a rate of over 50% (Aldemita andHodges 1996, Chan et al. 1993, Hiei et al. 1994).

Corn Transformation

Transformation of maize (Zea mays) is performed with a modification ofthe method described by Ishida et al. (1996) Nature Biotech 14(6):745-50. Transformation is genotype-dependent in corn and only specificgenotypes are amenable to transformation and regeneration. The inbredline A188 (University of Minnesota) or hybrids with A188 as a parent aregood sources of donor material for transformation, but other genotypescan be used successfully as well. Ears are harvested from corn plantapproximately 11 days after pollination (DAP) when the length of theimmature embryo is about 1 to 1.2 mm. Immature embryos are cocultivatedwith Agrobacterium tumefaciens containing the expression vector, andtransgenic plants are recovered through organogenesis. Excised embryosare grown on callus induction medium, then maize regeneration medium,containing the selection agent (for example imidazolinone but variousselection markers can be used). The Petri plates are incubated in thelight at 25° C. for 2-3 weeks, or until shoots develop. The green shootsare transferred from each embryo to maize rooting medium and incubatedat 25° C. for 2-3 weeks, until roots develop. The rooted shoots aretransplanted to soil in the greenhouse. T1 seeds are produced fromplants that exhibit tolerance to the selection agent and that contain asingle copy of the T-DNA insert.

Wheat Transformation

Transformation of wheat is performed with the method described by Ishidaet al. (1996) Nature Biotech 14(6): 745-50. The cultivar Bobwhite(available from CIMMYT, Mexico) is commonly used in transformation.Immature embryos are co-cultivated with Agrobacterium tumefacienscontaining the expression vector, and transgenic plants are recoveredthrough organogenesis. After incubation with Agrobacterium, the embryosare grown in vitro on callus induction medium, then regeneration medium,containing the selection agent (for example imidazolinone but variousselection markers can be used). The Petri plates are incubated in thelight at 25° C. for 2-3 weeks, or until shoots develop. The green shootsare transferred from each embryo to rooting medium and incubated at 25°C. for 2-3 weeks, until roots develop. The rooted shoots aretransplanted to soil in the greenhouse. T1 seeds are produced fromplants that exhibit tolerance to the selection agent and that contain asingle copy of the T-DNA insert.

Soybean Transformation

Soybean is transformed according to a modification of the methoddescribed in the Texas A&M U.S. Pat. No. 5,164,310. Several commercialsoybean varieties are amenable to transformation by this method. Thecultivar Jack (available from the Illinois Seed foundation) is commonlyused for transformation. Soybean seeds are sterilised for in vitrosowing. The hypocotyl, the radicle and one cotyledon are excised fromseven-day old young seedlings. The epicotyl and the remaining cotyledonare further grown to develop axillary nodes. These axillary nodes areexcised and incubated with Agrobacterium tumefaciens containing theexpression vector. After the cocultivation treatment, the explants arewashed and transferred to selection media. Regenerated shoots areexcised and placed on a shoot elongation medium. Shoots no longer than 1cm are placed on rooting medium until roots develop. The rooted shootsare transplanted to soil in the greenhouse. T1 seeds are produced fromplants that exhibit tolerance to the selection agent and that contain asingle copy of the T-DNA insert.

Rapeseed/Canola Transformation

Cotyledonary petioles and hypocotyls of 5-6 day old young seedling areused as explants for tissue culture and transformed according to Babicet al. (1998, Plant Cell Rep 17: 183-188). The commercial cultivarWestar (Agriculture Canada) is the standard variety used fortransformation, but other varieties can also be used. Canola seeds aresurface-sterilized for in vitro sowing. The cotyledon petiole explantswith the cotyledon attached are excised from the in vitro seedlings, andinoculated with Agrobacterium (containing the expression vector) bydipping the cut end of the petiole explant into the bacterialsuspension. The explants are then cultured for 2 days on MSBAP-3 mediumcontaining 3 mg/l BAP, 3% sucrose, 0.7% Phytagar at 23° C., 16 hr light.After two days of co-cultivation with Agrobacterium, the petioleexplants are transferred to MSBAP-3 medium containing 3 mg/l BAP,cefotaxime, carbenicillin, or timentin (300 mg/l) for 7 days, and thencultured on MSBAP-3 medium with cefotaxime, carbenicillin, or timentinand selection agent until shoot regeneration. When the shoots are 5-10mm in length, they are cut and transferred to shoot elongation medium(MSBAP-0.5, containing 0.5 mg/l BAP). Shoots of about 2 cm in length aretransferred to the rooting medium (MS0) for root induction. The rootedshoots are transplanted to soil in the greenhouse. T1 seeds are producedfrom plants that exhibit tolerance to the selection agent and thatcontain a single copy of the T-DNA insert.

Alfalfa Transformation

A regenerating clone of alfalfa (Medicago sativa) is transformed usingthe method of (McKersie et al., 1999 Plant Physiol 119: 839-847).Regeneration and transformation of alfalfa is genotype dependent andtherefore a regenerating plant is required. Methods to obtainregenerating plants have been described. For example, these can beselected from the cultivar Rangelander (Agriculture Canada) or any othercommercial alfalfa variety as described by Brown D C W and A Atanassov(1985. Plant Cell Tissue Organ Culture 4: 111-112). Alternatively, theRA3 variety (University of Wisconsin) has been selected for use intissue culture (Walker et al., 1978 Am J Bot 65:654-659). Petioleexplants are cocultivated with an overnight culture of Agrobacteriumtumefaciens C58C1 pMP90 (McKersie et al., 1999 Plant Physiol 119:839-847) or LBA4404 containing the expression vector. The explants arecocultivated for 3 d in the dark on SH induction medium containing 288mg/L Pro, 53 mg/L thioproline, 4.35 g/L K2S04, and 100 μmacetosyringinone. The explants are washed in half-strengthMurashige-Skoog medium (Murashige and Skoog, 1962) and plated on thesame SH induction medium without acetosyringinone but with a suitableselection agent and suitable antibiotic to inhibit Agrobacterium growth.After several weeks, somatic embryos are transferred to BOi2Ydevelopment medium containing no growth regulators, no antibiotics, and50 g/L sucrose. Somatic embryos are subsequently germinated onhalf-strength Murashige-Skoog medium. Rooted seedlings were transplantedinto pots and grown in a greenhouse. T1 seeds are produced from plantsthat exhibit tolerance to the selection agent and that contain a singlecopy of the T-DNA insert.

Example 17 Phenotypic Evaluation Procedure 17.1 Evaluation Setup

Approximately 35 independent T0 rice transformants were generated. Theprimary transformants were transferred from a tissue culture chamber toa greenhouse for growing and harvest of T1 seed. Six events, of whichthe T1 progeny segregated 3:1 for presence/absence of the transgene,were retained. For each of these events, approximately 10 T1 seedlingscontaining the transgene (hetero- and homo-zygotes) and approximately 10T1 seedlings lacking the transgene (nullizygotes) were selected bymonitoring visual marker expression. The transgenic plants and thecorresponding nullizygotes were grown side-by-side at random positions.Greenhouse conditions were of shorts days (12 hours light), 28° C. inthe light and 22° C. in the dark, and a relative humidity of 70%.

Four T1 events were further evaluated in the T2 generation following thesame evaluation procedure as for the T1 generation but with moreindividuals per event. From the stage of sowing until the stage ofmaturity the plants were passed several times through a digital imagingcabinet. At each time point digital images (2048×1536 pixels, 16 millioncolours) were taken of each plant from at least 6 different angles.

17.2 Statistical analysis: F-Test

A two factor ANOVA (analysis of variants) was used as a statisticalmodel for the overall evaluation of plant phenotypic characteristics. AnF-test was carried out on all the parameters measured of all the plantsof all the events transformed with the gene of the present invention.The F-test was carried out to check for an effect of the gene over allthe transformation events and to verify for an overall effect of thegene, also known as a global gene effect. The threshold for significancefor a true global gene effect was set at a 5% probability level for theF-test. A significant F-test value points to a gene effect, meaning thatit is not only the mere presence or position of the gene that is causingthe differences in phenotype.

To check for an effect of the genes within an event, i.e., for aline-specific effect, a t-test was performed within each event usingdata sets from the transgenic plants and the corresponding null plants.“Null plants” or “null segregants” or “nullizygotes” are the plantstreated in the same way as the transgenic plant, but from which thetransgene has segregated. Null plants can also be described as thehomozygous negative transformed plants. The threshold for significancefor the t-test is set at 10% probability level. The results for someevents can be above or below this threshold. This is based on thehypothesis that a gene might only have an effect in certain positions inthe genome, and that the occurrence of this position-dependent effect isnot uncommon. This kind of gene effect is also named herein a “lineeffect of the gene”. The p-value is obtained by comparing the t-value tothe t-distribution or alternatively, by comparing the F-value to theF-distribution. The p-value then gives the probability that the nullhypothesis (i.e., that there is no effect of the transgene) is correct.

17.3 Parameters Measured

From the stage of sowing until the stage of maturity the plants werepassed several times through a digital imaging cabinet. At each timepoint digital images (2048×1536 pixels, 16 million colours) were takenof each plant from at least 6 different angles.

The plant aboveground area (or leafy biomass) was determined by countingthe total number of pixels on the digital images from aboveground plantparts discriminated from the background. This value was averaged for thepictures taken on the same time point from the different angles and wasconverted to a physical surface value expressed in square mm bycalibration. Experiments show that the aboveground plant area measuredthis way correlates with the biomass of plant parts above ground. Theabove ground area is the area measured at the time point at which theplant had reached its maximal leafy biomass. The early vigour is theplant (seedling) aboveground area three weeks post-germination. Increasein root biomass is expressed as an increase in total root biomass(measured as maximum biomass of roots observed during the lifespan of aplant); or as an increase in the root/shoot index (measured as the ratiobetween root biomass and shoot biomass in the period of active growth ofroot and shoot).

Example 18 Results of the Phenotypic Evaluation of the Transgenic Plants

Upon analysis of the plants as described above, the inventors found thatplants transformed with the MADS15 gene construct had a higher rootyield, expressed as root/shoot index, compared to plants lacking theMADS15 transgene. The increase was 9.4% (p-value 0.0207) in T1 and 25.7%(p-value 0.0002) in T2. The p-values show that the increases weresignificant.

Example Section C MADS15 Down-Regulated

See also Examples 8 to 13 for the identification and characterisation ofMADS15 related sequences.

Example 19 Gene Cloning

The Oryza sativa MADS15 gene was amplified by PCR using as template anOryza sativa seedling cDNA library (Invitrogen, Paisley, UK). Afterreverse transcription of RNA extracted from seedlings, the cDNAs werecloned into pCMV Sport 6.0. Average insert size of the bank was 1.5 kband the original number of clones was of the order of 1.59×10⁷ cfu.Original titer was determined to be 9.6×10⁵ cfu/ml after firstamplification of 6×10¹¹ cfu/ml. After plasmid extraction, 200 ng oftemplate was used in a 50 μl PCR mix. Primers prm06892 (SEQ ID NO: 111;sense, start codon in bold, AttB1 site in italic:-ggggacaagtttgtacaaaaaagcaggcttaaacaatggg cgggggaaggt-3′) and prm06893(SEQ ID NO: 112; reverse, complementary, AttB2 site in italic:5′-ggggaccactttgtacaagaaagctgggtttggccgacgacgacgac-3′), which includethe AttB sites for Gateway recombination, were used for PCRamplification. PCR was performed using Hifi Taq DNA polymerase instandard conditions. A PCR fragment of around 915 bp was amplified andpurified also using standard methods. The PCR fragment was subsequentlyused for the preparation of a hairpin construct, using techniques knownin the art. The first step of the Gateway procedure, the BP reaction,was then performed, during which the hairpin construct recombines invivo with the pDONR201 plasmid to produce, according to the Gatewayterminology, an “entry clone”, pMADS15hp. Plasmid pDONR201 was purchasedfrom Invitrogen, as part of the Gateway® technology.

Example 20 Vector Construction

The entry clone pMADS15hp was subsequently used in an LR reaction withp01519, a destination vector for the inverted repeat construct. Thisvector contains as functional elements within the T-DNA borders: a plantselectable marker; a screenable marker expression cassette; and aGateway cassette intended for LR in vivo recombination such that thesequence of interest from the entry clone is integrated as an invertedrepeat. A rice GOS2 promoter (SEQ ID NO: 174, alternatively, SEQ ID NO:113 is equally useful) for constitutive expression was located upstreamof this Gateway cassette.

After the LR recombination step, the resulting expression vector withthe inverted repeat, FIG. 10) were transformed into Agrobacterium strainLBA4044 and subsequently to Oryza sativa plants.

Example 21 Plant Transformation Rice Transformation

The Agrobacterium containing the expression vector was used to transformOryza sativa plants. Mature dry seeds of the rice japonica cultivarNipponbare were dehusked. Sterilization was carried out by incubatingfor one minute in 70% ethanol, followed by 30 minutes in 0.2% HgCl₂,followed by a 6 times 15 minutes wash with sterile distilled water. Thesterile seeds were then germinated on a medium containing 2,4-D (callusinduction medium). After incubation in the dark for four weeks,embryogenic, scutellum-derived calli were excised and propagated on thesame medium. After two weeks, the calli were multiplied or propagated bysubculture on the same medium for another 2 weeks. Embryogenic calluspieces were sub-cultured on fresh medium 3 days before co-cultivation(to boost cell division activity).

Agrobacterium strain LBA4404 containing the expression vector was usedfor co-cultivation. Agrobacterium was inoculated on AB medium with theappropriate antibiotics and cultured for 3 days at 28° C. The bacteriawere then collected and suspended in liquid co-cultivation medium to adensity (OD₆₀₀) of about 1. The suspension was then transferred to aPetri dish and the calli immersed in the suspension for 15 minutes. Thecallus tissues were then blotted dry on a filter paper and transferredto solidified, co-cultivation medium and incubated for 3 days in thedark at 25° C. Co-cultivated calli were grown on 2,4-D-containing mediumfor 4 weeks in the dark at 28° C. in the presence of a selection agent.During this period, rapidly growing resistant callus islands developed.After transfer of this material to a regeneration medium and incubationin the light, the embryogenic potential was released and shootsdeveloped in the next four to five weeks. Shoots were excised from thecalli and incubated for 2 to 3 weeks on an auxin-containing medium fromwhich they were transferred to soil. Hardened shoots were grown underhigh humidity and short days in a greenhouse.

The primary transformants were transferred from a tissue culture chamberto a greenhouse. After a quantitative PCR analysis to verify copy numberof the T-DNA insert, only single copy transgenic plants that exhibittolerance to the selection agent were kept for harvest of T1 seed. Seedswere then harvested three to five months after transplanting. The methodyielded single locus transformants at a rate of over 50% (Aldemita andHodges 1996, Chan et al. 1993, Hiei et al. 1994).

Corn Transformation

Transformation of maize (Zea mays) is performed with a modification ofthe method described by Ishida et al. (1996) Nature Biotech 14(6):745-50. Transformation is genotype-dependent in corn and only specificgenotypes are amenable to transformation and regeneration. The inbredline A188 (University of Minnesota) or hybrids with A188 as a parent aregood sources of donor material for transformation, but other genotypescan be used successfully as well. Ears are harvested from corn plantapproximately 11 days after pollination (DAP) when the length of theimmature embryo is about 1 to 1.2 mm. Immature embryos are cocultivatedwith Agrobacterium tumefaciens containing the expression vector, andtransgenic plants are recovered through organogenesis. Excised embryosare grown on callus induction medium, then maize regeneration medium,containing the selection agent (for example imidazolinone but variousselection markers can be used). The Petri plates are incubated in thelight at 25° C. for 2-3 weeks, or until shoots develop. The green shootsare transferred from each embryo to maize rooting medium and incubatedat 25° C. for 2-3 weeks, until roots develop. The rooted shoots aretransplanted to soil in the greenhouse. T1 seeds are produced fromplants that exhibit tolerance to the selection agent and that contain asingle copy of the T-DNA insert.

Wheat Transformation

Transformation of wheat is performed with the method described by Ishidaet al. (1996) Nature Biotech 14(6): 745-50. The cultivar Bobwhite(available from CIMMYT, Mexico) is commonly used in transformation.Immature embryos are co-cultivated with Agrobacterium tumefacienscontaining the expression vector, and transgenic plants are recoveredthrough organogenesis. After incubation with Agrobacterium, the embryosare grown in vitro on callus induction medium, then regeneration medium,containing the selection agent (for example imidazolinone but variousselection markers can be used). The Petri plates are incubated in thelight at 25° C. for 2-3 weeks, or until shoots develop. The green shootsare transferred from each embryo to rooting medium and incubated at 25°C. for 2-3 weeks, until roots develop. The rooted shoots aretransplanted to soil in the greenhouse. T1 seeds are produced fromplants that exhibit tolerance to the selection agent and that contain asingle copy of the T-DNA insert.

Soybean Transformation

Soybean is transformed according to a modification of the methoddescribed in the Texas A&M U.S. Pat. No. 5,164,310. Several commercialsoybean varieties are amenable to transformation by this method. Thecultivar Jack (available from the Illinois Seed foundation) is commonlyused for transformation. Soybean seeds are sterilised for in vitrosowing. The hypocotyl, the radicle and one cotyledon are excised fromseven-day old young seedlings. The epicotyl and the remaining cotyledonare further grown to develop axillary nodes. These axillary nodes areexcised and incubated with Agrobacterium tumefaciens containing theexpression vector. After the cocultivation treatment, the explants arewashed and transferred to selection media. Regenerated shoots areexcised and placed on a shoot elongation medium. Shoots no longer than 1cm are placed on rooting medium until roots develop. The rooted shootsare transplanted to soil in the greenhouse. T1 seeds are produced fromplants that exhibit tolerance to the selection agent and that contain asingle copy of the T-DNA insert.

Rapeseed/Canola Transformation

Cotyledonary petioles and hypocotyls of 5-6 day old young seedling areused as explants for tissue culture and transformed according to Babicet al. (1998, Plant Cell Rep 17: 183-188). The commercial cultivarWestar (Agriculture Canada) is the standard variety used fortransformation, but other varieties can also be used. Canola seeds aresurface-sterilized for in vitro sowing. The cotyledon petiole explantswith the cotyledon attached are excised from the in vitro seedlings, andinoculated with Agrobacterium (containing the expression vector) bydipping the cut end of the petiole explant into the bacterialsuspension. The explants are then cultured for 2 days on MSBAP-3 mediumcontaining 3 mg/l BAP, 3% sucrose, 0.7% Phytagar at 23° C., 16 hr light.After two days of co-cultivation with Agrobacterium, the petioleexplants are transferred to MSBAP-3 medium containing 3 mg/l BAP,cefotaxime, carbenicillin, or timentin (300 mg/l) for 7 days, and thencultured on MSBAP-3 medium with cefotaxime, carbenicillin, or timentinand selection agent until shoot regeneration. When the shoots are 5-10mm in length, they are cut and transferred to shoot elongation medium(MSBAP-0.5, containing 0.5 mg/l BAP). Shoots of about 2 cm in length aretransferred to the rooting medium (MS0) for root induction. The rootedshoots are transplanted to soil in the greenhouse. T1 seeds are producedfrom plants that exhibit tolerance to the selection agent and thatcontain a single copy of the T-DNA insert.

Alfalfa Transformation

A regenerating clone of alfalfa (Medicago sativa) is transformed usingthe method of (McKersie et al., 1999 Plant Physiol 119: 839-847).Regeneration and transformation of alfalfa is genotype dependent andtherefore a regenerating plant is required. Methods to obtainregenerating plants have been described. For example, these can beselected from the cultivar Rangelander (Agriculture Canada) or any othercommercial alfalfa variety as described by Brown D C W and A Atanassov(1985. Plant Cell Tissue Organ Culture 4: 111-112). Alternatively, theRA3 variety (University of Wisconsin) has been selected for use intissue culture (Walker et al., 1978 Am J Bot 65:654-659). Petioleexplants are cocultivated with an overnight culture of Agrobacteriumtumefaciens C58C1 pMP90 (McKersie et al., 1999 Plant Physiol 119:839-847) or LBA4404 containing the expression vector. The explants arecocultivated for 3 d in the dark on SH induction medium containing 288mg/L Pro, 53 mg/L thioproline, 4.35 g/L K2SO4, and 100 μmacetosyringinone. The explants are washed in half-strengthMurashige-Skoog medium (Murashige and Skoog, 1962) and plated on thesame SH induction medium without acetosyringinone but with a suitableselection agent and suitable antibiotic to inhibit Agrobacterium growth.After several weeks, somatic embryos are transferred to BOi2Ydevelopment medium containing no growth regulators, no antibiotics, and50 g/L sucrose. Somatic embryos are subsequently germinated onhalf-strength Murashige-Skoog medium. Rooted seedlings were transplantedinto pots and grown in a greenhouse. T1 seeds are produced from plantsthat exhibit tolerance to the selection agent and that contain a singlecopy of the T-DNA insert.

Example 22 Evaluation Methods of Plants Transformed with the MADS15Inverted Repeat Under Control of the Rice GOS2 Promoter

Approximately 15 to 20 independent T0 rice transformants were generated.The primary transformants were transferred from a tissue culture chamberto a greenhouse for growing and harvest of T1 seed. Eight events for theinverted repeat construct of which the T1 progeny segregated 3:1 forpresence/absence of the transgene, were retained. For each of theseevents, approximately 10 T1 seedlings containing the transgene (hetero-and homozygotes) and approximately 10 T1 seedlings lacking the transgene(nullizygotes) were selected by monitoring visual marker expression. Theselected T1 plants were transferred to a greenhouse. Each plant receiveda unique barcode label to link unambiguously the phenotyping data to thecorresponding plant. The selected T1 plants were grown on soil in 10 cmdiameter pots under the following environmental settings:photoperiod=11.5 h, daylight intensity=30,000 lux or more, daytimetemperature=28° C. or higher, night time temperature=22° C., relativehumidity=60-70%. Transgenic plants and the corresponding nullizygoteswere grown side-by-side at random positions. From the stage of sowinguntil the stage of maturity the plants were passed several times througha digital imaging cabinet. At each time point digital images (2048×1536pixels, 16 million colours) were taken of each plant from at least 6different angles.

The plant aboveground area (or leafy biomass) was determined by countingthe total number of pixels on the digital images from aboveground plantparts discriminated from the background. This value was averaged for thepictures taken on the same time point from the different angles and wasconverted to a physical surface value expressed in square mm bycalibration. Experiments show that the aboveground plant area measuredthis way correlates with the biomass of plant parts above ground. TheAreamax is the above ground area at the time point at which the planthad reached its maximal leafy biomass.

The mature primary panicles were harvested, bagged, barcode-labelled andthen dried for three days in the oven at 37° C. The panicles were thenthreshed and all the seeds collected. The filled husks were separatedfrom the empty ones using an air-blowing device. After separation, bothseed lots were then counted using a commercially available countingmachine. The empty husks were discarded. The filled husks were weighedon an analytical balance and the cross-sectional area of the seeds wasmeasured using digital imaging. This procedure resulted in the set ofthe following seed-related parameters:

The flowers-per-panicle is a parameter estimating the average number offlorets per panicle on a plant, derived from the number of total seedsdivided by the number of first panicles. The tallest panicle and all thepanicles that overlapped with the tallest panicle when alignedvertically, were considered as first panicles and were counted manually.The number of filled seeds was determined by counting the number offilled husks that remained after the separation step. The total seedyield (total seed weight) was measured by weighing all filled husksharvested from a plant. Total seed number per plant was measured bycounting the number of husks harvested from a plant and corresponds tothe number of florets per plant. These parameters were derived in anautomated way from the digital images using image analysis software andwere analysed statistically. Individual seed parameters (includingwidth, length, area, weight) were measured using a custom-made deviceconsisting of two main components, a weighing and imaging device,coupled to software for image analysis.

A two factor ANOVA (analyses of variance) corrected for the unbalanceddesign was used as statistical model for the overall evaluation of plantphenotypic characteristics. An F-test was carried out on all theparameters measured of all the plants of all the events transformed withthat gene. The F-test was carried out to check for an effect of the geneover all the transformation events and to verify for an overall effectof the gene, also named herein “global gene effect”. If the value of theF test shows that the data are significant, than it is concluded thatthere is a “gene” effect, meaning that not only presence or the positionof the gene is causing the effect. The threshold for significance for atrue global gene effect is set at 5% probability level for the F test.

To check for an effect of the genes within an event, i.e., for aline-specific effect, a t-test was performed within each event usingdata sets from the transgenic plants and the corresponding null plants.“Null plants” or “null segregants” or “nullizygotes” are the plantstreated in the same way as the transgenic plant, but from which thetransgene has segregated. Null plants can also be described as thehomozygous negative transformed plants. The threshold for significancefor the t-test is set at 10% probability level. The results for someevents can be above or below this threshold. This is based on thehypothesis that a gene might only have an effect in certain positions inthe genome, and that the occurrence of this position-dependent effect isnot uncommon. This kind of gene effect is also named herein a “lineeffect of the gene”. The p-value is obtained by comparing the t-value tothe t-distribution or alternatively, by comparing the F-value to theF-distribution. The p-value then gives the probability that the nullhypothesis (i.e., that there is no effect of the transgene) is correct.

Example 23 Measurement of Yield-Related Parameters for the InvertedRepeat Construct Transformants

Upon analysis of the seeds as described above, the inventors found thatplants transformed with the hairpin MADS15 gene construct had a higherseed yield, expressed as number of filled seeds, total weight of seeds,total number of seeds, and flowers per panicle, compared to plantslacking the MADS15 transgene, whereas the plants transformed with thesense MADS15 gene construct showed opposite effects. The p-values showthat the increases were significant.

The results obtained for plants in the T1 generation are summarised inTable H, which represent the mean values for all the tested lines:

TABLE H % difference p-value number of filled seeds +122 0.0000 totalweight of seeds +112 0.0000 total number of seeds +27 0.0000 flowers perpanicle +25 0.0000

Example Section D PLT Example 24 Identification of Sequences Related tothe Nucleic Acid Sequence Used in the Methods of the Invention

Sequences (full length cDNA, ESTs or genomic) related to the nucleicacid sequence used in the methods of the present invention wereidentified amongst those maintained in the Entrez Nucleotides databaseat the National Center for Biotechnology Information (NCBI) usingdatabase sequence search tools, such as the Basic Local Alignment Tool(BLAST) (Altschul et al. (1990) J. Mol. Biol. 215:403-410; and Altschulet al. (1997) Nucleic Acids Res. 25:3389-3402). The program is used tofind regions of local similarity between sequences by comparing nucleicacid or polypeptide sequences to sequence databases and by calculatingthe statistical significance of matches. For example, the polypeptideencoded by the nucleic acid of the present invention was used for theTBLASTN algorithm, with default settings and the filter to ignore lowcomplexity sequences set off. The output of the analysis was viewed bypairwise comparison, and ranked according to the probability score(E-value), where the score reflect the probability that a particularalignment occurs by chance (the lower the E-value, the more significantthe hit). In addition to E-values, comparisons were also scored bypercentage identity. Percentage identity refers to the number ofidentical nucleotides (or amino acids) between the two compared nucleicacid (or polypeptide) sequences over a particular length. In someinstances, the default parameters may be adjusted to modify thestringency of the search. For example the E-value may be increased toshow less stringent matches. This way, short nearly exact matches may beidentified.

The Table I below provides a list of nucleic acid sequences related tothe nucleic acid sequence used in the methods of the present invention.

TABLE I nucleic acid sequences related to the nucleic acid sequence usedin the methods of the present invention Nucleic acid Polypeptide NCBIaccession Name SEQ ID NO SEQ ID NO number Source organism Arath_PLT1 175176 full length NM_112975 Arabidopsis thaliana (At3g20840) Arath_PLT2177 178 full length NM_103997 Arabidopsis thaliana (At1g51190) Glyma_PLT179 180 full length BU964973.1 Glycine max CA783156.1 BM309051.1BM309377.1 Glyma_PLT2 181 182 full length BU926204.1 Glycine maxBU547204.1 CA783156.1 BU927164.1 Medtr_PLT 183 184 full lengthAC144930.20 Medicago truncatula Orysa_PLT 185 186 full length NM_190301Oryza sativa Zeama_PLT 187 188 full length CS155772.1 Zea mays Lotco_PLT189 190 partial AP007400 Lotus corniculatus Poptr_PLT I 199 200 fulllength scaff_III.1595 Populus tremuloides Poptr_PLT II 201 202 fulllength scaff_I.328 Populus tremuloides Vitvi_PLT 203 204 partialAM469514 Vitis vinifera Brana_PLT 205 206 partial CN730825 Brassicanapus Phaco_PLT 207 208 partial CA902624.1| Phaseolus coccineus

In some instances, related sequences have tentatively been assembled andpublicly disclosed by research institutions, such as The Institute forGenomic Research (TIGR). The Eukaryotic Gene Orthologs (EGO) databasemay be used to identify such related sequences, either by keyword searchor by using the BLAST algorithm with the nucleic acid or polypeptidesequence of interest.

Example 25 Cloning of the Nucleic Acid Sequences Used in the Methods ofthe Invention

The nucleic acid sequences used in the methods of the invention wereamplified by PCR using as template a custom-made Arabidopsis thalianacDNA library made starting from RNA extracted from different tissues (inpCMV Sport 6.0; Invitrogen, Paisley, UK). PCR was performed using HifiTaq DNA polymerase in standard conditions, using 200 ng of template in a50 μl PCR mix. The primers used to amplify SEQ ID NO: 175 (PLT1) wereprm08180 (SEQ ID NO: 195; sense, start codon in bold, AttB1 site initalic: 5′ GGGGACAAGTTTGTA CAAAAAAGCAGGCTTAAACAATGATCAATCCACACGGTG 3′)and prm08181 (SEQ ID NO: 196; reverse, complementary, AttB2 site initalic: 5′ GGGGACCACTTTGTACAAGAAAG CTGGGTTCCTTGTTTACTCATTCCACA 3′),which include the AttB sites for Gateway recombination. The primers usedto amplify SEQ ID NO: 177 (PLT2) were prm08182 (SEQ ID NO: 197; sense,start codon in bold, AttB1 site in italic:5′GGGGACAAGTTTGTACAAAAAAGCAGGCTTAAACAATGAATTCTAACA ACTGGCTC 3′) andprm08183 (SEQ ID NO: 198; reverse, complementary, AttB2 site in italic:5′ GGGGACCACTTTGTACAAGAAAGCTGGGTTCATCTTTTATTCATTCCACA 3′),

The amplified PCR fragments were purified also using standard methods.The first step of the Gateway procedure, the BP reaction, was thenperformed, during which the PCR fragment recombines in vivo with thepDONR201 plasmid to produce, according to the Gateway terminology, an“entry clone”, pPLT1 for SEQ ID NO: 175 and pPLT2 for SEQ ID NO: 177.Plasmid pDONR201 was purchased from Invitrogen, as part of the Gateway®technology.

Example 26 Expression Vector Construction

The entry clones pPLT1 and pPLT2 were subsequently used in an LRreaction with destination vectors used for Oryza sativa transformation.A first vector contains as functional elements within the T-DNA borders:a plant selectable marker; a screenable marker expression cassette; anda Gateway cassette intended for LR in vivo recombination with thenucleic acid sequence of interest already cloned in the entry clone. Arice constitutive promoter, a GOS2 promoter (SEQ ID NO: 210,alternatively SEQ ID NO: 194 is equally useful) was located upstream ofthis Gateway cassette.

A second vector contains the same functional elements within the T-DNAborders: a plant selectable marker; a screenable marker expressioncassette; and a Gateway cassette intended for LR in vivo recombinationwith the nucleic acid sequence of interest already cloned in the entryclone. A rice promoter for expression in meristems, an MT promoter (SEQID NO: 211) (PRO0126) was located upstream of this Gateway cassette.

After the LR recombination step, the resulting expression vectors,pGOS2::PLT1, pGOS2; PLT2, pMT::PLT1 and pMT::PLT2 (FIG. 16 shows theconstruct with the GOS2 promoter) were independently transformed intoAgrobacterium strain LBA4044 and subsequently to Oryza sativa plants.Transformed rice plants were allowed to grow and were then examined forthe parameters described below.

Example 27 Crop Transformation

The transformed Agrobacterium containing the expression vectors wereused independently to transform Oryza sativa plants. Mature dry seeds ofthe rice japonica cultivar Nipponbare were dehusked. Sterilization wascarried out by incubating for one minute in 70% ethanol, followed by 30minutes in 0.2% HgCl₂, followed by a 6 times 15 minutes wash withsterile distilled water. The sterile seeds were then germinated on amedium containing 2,4-D (callus induction medium). After incubation inthe dark for four weeks, embryogenic, scutellum-derived calli wereexcised and propagated on the same medium. After two weeks, the calliwere multiplied or propagated by subculture on the same medium foranother 2 weeks. Embryogenic callus pieces were sub-cultured on freshmedium 3 days before co-cultivation (to boost cell division activity).

Agrobacterium strain LBA4404 containing the expression vector was usedfor co-cultivation. Agrobacterium was inoculated on AB medium with theappropriate antibiotics and cultured for 3 days at 28° C. The bacteriawere then collected and suspended in liquid co-cultivation medium to adensity (OD₆₀₀) of about 1. The suspension was then transferred to aPetri dish and the calli immersed in the suspension for 15 minutes. Thecallus tissues were then blotted dry on a filter paper and transferredto solidified, co-cultivation medium and incubated for 3 days in thedark at 25° C. Co-cultivated calli were grown on 2,4-D-containing mediumfor 4 weeks in the dark at 28° C. in the presence of a selection agent.During this period, rapidly growing resistant callus islands developed.After transfer of this material to a regeneration medium and incubationin the light, the embryogenic potential was released and shootsdeveloped in the next four to five weeks. Shoots were excised from thecalli and incubated for 2 to 3 weeks on an auxin-containing medium fromwhich they were transferred to soil. Hardened shoots were grown underhigh humidity and short days in a greenhouse.

Approximately 35 independent T0 rice transformants were generated forone construct. The primary transformants were transferred from a tissueculture chamber to a greenhouse. After a quantitative PCR analysis toverify copy number of the T-DNA insert, only single copy transgenicplants that exhibit tolerance to the selection agent were kept forharvest of T1 seed. Seeds were then harvested three to five months aftertransplanting. The method yielded single locus transformants at a rateof over 50% (Aldemita and Hodges 1996, Chan et al. 1993, Hiei et al.1994).

Corn Transformation

Transformation of maize (Zea mays) is performed with a modification ofthe method described by Ishida et al. (1996) Nature Biotech 14(6):745-50. Transformation is genotype-dependent in corn and only specificgenotypes are amenable to transformation and regeneration. The inbredline A188 (University of Minnesota) or hybrids with A188 as a parent aregood sources of donor material for transformation, but other genotypescan be used successfully as well. Ears are harvested from corn plantapproximately 11 days after pollination (DAP) when the length of theimmature embryo is about 1 to 1.2 mm. Immature embryos are cocultivatedwith Agrobacterium tumefaciens containing the expression vector, andtransgenic plants are recovered through organogenesis. Excised embryosare grown on callus induction medium, then maize regeneration medium,containing the selection agent (for example imidazolinone but variousselection markers can be used). The Petri plates are incubated in thelight at 25° C. for 2-3 weeks, or until shoots develop. The green shootsare transferred from each embryo to maize rooting medium and incubatedat 25° C. for 2-3 weeks, until roots develop. The rooted shoots aretransplanted to soil in the greenhouse. T1 seeds are produced fromplants that exhibit tolerance to the selection agent and that contain asingle copy of the T-DNA insert.

Wheat Transformation

Transformation of wheat is performed with the method described by Ishidaet al. (1996) Nature Biotech 14(6): 745-50. The cultivar Bobwhite(available from CIMMYT, Mexico) is commonly used in transformation.Immature embryos are co-cultivated with Agrobacterium tumefacienscontaining the expression vector, and transgenic plants are recoveredthrough organogenesis. After incubation with Agrobacterium, the embryosare grown in vitro on callus induction medium, then regeneration medium,containing the selection agent (for example imidazolinone but variousselection markers can be used). The Petri plates are incubated in thelight at 25° C. for 2-3 weeks, or until shoots develop. The green shootsare transferred from each embryo to rooting medium and incubated at 25°C. for 2-3 weeks, until roots develop. The rooted shoots aretransplanted to soil in the greenhouse. T1 seeds are produced fromplants that exhibit tolerance to the selection agent and that contain asingle copy of the T-DNA insert.

Soybean Transformation

Soybean is transformed according to a modification of the methoddescribed in the Texas A&M U.S. Pat. No. 5,164,310. Several commercialsoybean varieties are amenable to transformation by this method. Thecultivar Jack (available from the Illinois Seed foundation) is commonlyused for transformation. Soybean seeds are sterilised for in vitrosowing. The hypocotyl, the radicle and one cotyledon are excised fromseven-day old young seedlings. The epicotyl and the remaining cotyledonare further grown to develop axillary nodes. These axillary nodes areexcised and incubated with Agrobacterium tumefaciens containing theexpression vector. After the cocultivation treatment, the explants arewashed and transferred to selection media. Regenerated shoots areexcised and placed on a shoot elongation medium. Shoots no longer than 1cm are placed on rooting medium until roots develop. The rooted shootsare transplanted to soil in the greenhouse. T1 seeds are produced fromplants that exhibit tolerance to the selection agent and that contain asingle copy of the T-DNA insert.

Rapeseed/Canola Transformation

Cotyledonary petioles and hypocotyls of 5-6 day old young seedling areused as explants for tissue culture and transformed according to Babicet al. (1998, Plant Cell Rep 17: 183-188). The commercial cultivarWestar (Agriculture Canada) is the standard variety used fortransformation, but other varieties can also be used. Canola seeds aresurface-sterilized for in vitro sowing. The cotyledon petiole explantswith the cotyledon attached are excised from the in vitro seedlings, andinoculated with Agrobacterium (containing the expression vector) bydipping the cut end of the petiole explant into the bacterialsuspension. The explants are then cultured for 2 days on MSBAP-3 mediumcontaining 3 mg/l BAP, 3% sucrose, 0.7% Phytagar at 23° C., 16 hr light.After two days of co-cultivation with Agrobacterium, the petioleexplants are transferred to MSBAP-3 medium containing 3 mg/l BAP,cefotaxime, carbenicillin, or timentin (300 mg/l) for 7 days, and thencultured on MSBAP-3 medium with cefotaxime, carbenicillin, or timentinand selection agent until shoot regeneration. When the shoots are 5-10mm in length, they are cut and transferred to shoot elongation medium(MSBAP-0.5, containing 0.5 mg/l BAP). Shoots of about 2 cm in length aretransferred to the rooting medium (MS0) for root induction. The rootedshoots are transplanted to soil in the greenhouse. T1 seeds are producedfrom plants that exhibit tolerance to the selection agent and thatcontain a single copy of the T-DNA insert.

Alfalfa Transformation

A regenerating clone of alfalfa (Medicago sativa) is transformed usingthe method of (McKersie et al., 1999 Plant Physiol 119: 839-847).Regeneration and transformation of alfalfa is genotype dependent andtherefore a regenerating plant is required. Methods to obtainregenerating plants have been described. For example, these can beselected from the cultivar Rangelander (Agriculture Canada) or any othercommercial alfalfa variety as described by Brown D C W and A Atanassov(1985. Plant Cell Tissue Organ Culture 4: 111-112). Alternatively, theRA3 variety (University of Wisconsin) has been selected for use intissue culture (Walker et al., 1978 Am J Bot 65:654-659). Petioleexplants are cocultivated with an overnight culture of Agrobacteriumtumefaciens C58C1 pMP90 (McKersie et al., 1999 Plant Physiol 119:839-847) or LBA4404 containing the expression vector. The explants arecocultivated for 3 d in the dark on SH induction medium containing 288mg/L Pro, 53 mg/L thioproline, 4.35 g/L K₂SO₄, and 100 μmacetosyringinone. The explants are washed in half-strengthMurashige-Skoog medium (Murashige and Skoog, 1962) and plated on thesame SH induction medium without acetosyringinone but with a suitableselection agent and suitable antibiotic to inhibit Agrobacterium growth.After several weeks, somatic embryos are transferred to BOi2Ydevelopment medium containing no growth regulators, no antibiotics, and50 g/L sucrose. Somatic embryos are subsequently germinated onhalf-strength Murashige-Skoog medium. Rooted seedlings were transplantedinto pots and grown in a greenhouse. T1 seeds are produced from plantsthat exhibit tolerance to the selection agent and that contain a singlecopy of the T-DNA insert.

Example 28 Phenotypic Evaluation Procedure 28.1 Evaluation SetupEvaluation in Normal Growth Conditions

The primary transformants were transferred from a tissue culture chamberto a greenhouse for growing and harvest of T1 seed. Six events, of whichthe T1 progeny segregated 3:1 for presence/absence of the transgene,were retained. For each of these events, approximately 10 T1 seedlingscontaining the transgene (hetero- and homo-zygotes) and approximately 10T1 seedlings lacking the transgene (nullizygotes) were selected bymonitoring visual marker expression. The transgenic plants and thecorresponding nullizygotes were grown side-by-side at random positions.Greenhouse conditions were of shorts days (12 hours light), 28° C. inthe light and 22° C. in the dark, and a relative humidity of 70%.

Evaluation Under Reduced Nitrogen Availability

The rice plants were grown in potting soil as under normal conditionsexcept for the nutrient solution. The pots were watered fromtransplantation to maturation with a specific nutrient solutioncontaining reduced N nitrogen (N) content, usually between 7 to 8 timesless. The rest of the cultivation (plant maturation, seed harvest) wasthe same as for plants not grown under abiotic stress.

28.2 Statistical Analysis: F-Test

A two factor ANOVA (analysis of variants) was used as a statisticalmodel for the overall evaluation of plant phenotypic characteristics. AnF-test was carried out on all the parameters measured of all the plantsof all the events transformed with the gene of the present invention.The F-test was carried out to check for an effect of the gene over allthe transformation events and to verify for an overall effect of thegene, also known as a global gene effect. The threshold for significancefor a true global gene effect was set at a 5% probability level for theF-test. A significant F-test value points to a gene effect, meaning thatit is not only the mere presence or position of the gene that is causingthe differences in phenotype.

To check for an effect of the genes within an event, i.e., for aline-specific effect, a t-test was performed within each event usingdata sets from the transgenic plants and the corresponding null plants.“Null plants” or “null segregants” or “nullizygotes” are the plantstreated in the same way as the transgenic plant, but from which thetransgene has segregated. Null plants can also be described as thehomozygous negative transformed plants. The threshold for significancefor the t-test is set at 10% probability level. The results for someevents can be above or below this threshold. This is based on thehypothesis that a gene might only have an effect in certain positions inthe genome, and that the occurrence of this position-dependent effect isnot uncommon. This kind of gene effect is also named herein a “lineeffect of the gene”. The p-value is obtained by comparing the t-value tothe t-distribution or alternatively, by comparing the F-value to theF-distribution. The p-value then gives the probability that the nullhypothesis (i.e., that there is no effect of the transgene) is correct.

28.3 Parameters Measured Biomass-Related Parameter Measurement

From the stage of sowing until the stage of maturity the plants werepassed several times through a digital imaging cabinet. At each timepoint digital images (2048×1536 pixels, 16 million colours) were takenof each plant from at least 6 different angles.

The plant aboveground area (or leafy biomass, areamax) was determined bycounting the total number of pixels on the digital images fromaboveground plant parts discriminated from the background. This valuewas averaged for the pictures taken on the same time point from thedifferent angles and was converted to a physical surface value expressedin square mm by calibration. Experiments show that the aboveground plantarea measured this way correlates with the biomass of plant parts aboveground. The above ground area is the area measured at the time point atwhich the plant had reached its maximal leafy biomass. The early vigouris the plant (seedling) aboveground area three weeks post-germination.Increase in root biomass is expressed as an increase in total rootbiomass (measured as maximum biomass of roots observed during thelifespan of a plant); or as an increase in the root/shoot index(measured as the ratio between root mass and shoot mass in the period ofactive growth of root and shoot).

Seed-Related Parameter Measurements

The mature primary panicles were harvested, counted, bagged,barcode-labelled and then dried for three days in an oven at 37° C. Thepanicles were then threshed and all the seeds were collected andcounted. The filled husks were separated from the empty ones using anair-blowing device. The empty husks were discarded and the remainingfraction was counted again. The filled husks were weighed on ananalytical balance. The number of filled seeds was determined bycounting the number of filled husks that remained after the separationstep. The total seed yield was measured by weighing all filled husksharvested from a plant. Total seed number per plant was measured bycounting the number of husks harvested from a plant. Thousand KernelWeight (TKW) is extrapolated from the number of filled seeds counted andtheir total weight. The Harvest Index (HI) in the present invention isdefined as the ratio between the total seed yield and the above groundarea (mm²), multiplied by a factor 10⁶. The total number of flowers perpanicle as defined in the present invention is the ratio between thetotal number of seeds and the number of mature primary panicles. Theseed fill rate as defined in the present invention is the proportion(expressed as a %) of the number of filled seeds over the total numberof seeds (or florets).

Example 29 Results of the Phenotypic Evaluation of the Transgenic Plants29.1 Results of the Phenotypic Evaluation of the Transgenics PlantsGrown Under Normal Growth Conditions, Expressing Either PLT1 or PLT2Nucleic Acid Sequence Under the Control of a Constitutive Promoter

The TKW measurement results of the T1 seeds of PLT1 and PLT2 transgenicrice plants grown under normal growth conditions, are shown in Table J,in absolute values (averaged events) and as a percentage compared towild type plants. A substantial increase in TKW is observed for thetransgenic seeds containing either construct, compared to wild typeseeds.

TABLE J Results of TKW measurements of the T1 seeds of PLT1 and PLT2transgenic plants grown under normal growth conditions. TKW (g) %increase PLT1 transgenics 0.0290 16% PLT2 transgenics 0.0288 15% WT0.0250

Individual seed parameters (including width, length, area, weight) weremeasured using a custom-made device consisting of two main components, aweighing and imaging device, coupled to software for image analysis.

The seed area and seed length measurement results of T1 seeds of PLT1and PLT2 transgenic rice plants are shown in Table K in absolute values(averaged events) and as a percentage compared to wild type plants. Aclear increase in both seed area and seed length is observed for thetransgenic seeds containing either construct, compared to wild typeseeds.

TABLE K Results of seed area and seed length measurements of the T1seeds of PLT1 and PLT2 transgenic plants grown under normal growthconditions. Seed area Seed length (mm²) % increase (mm) % increase PLT1transgenics 27.60 12% 9.4 13% PLT2 transgenics 27.35 11% 9.4 13% WT24.56 8.3

Seed width was not significantly affected on the T1 seeds of the PLT andPLT2 transgenic plants (data not shown).

29.2 Results of the Phenotypic Evaluation of the Transgenics PlantsGrown Under Reduced Nitrogen Availability Conditions, Expressing EitherPLT1 or PLT2 Nucleic Acid Sequence Under the Control of a ConstitutivePromoter

The TKW measurement results of the T1 seeds of PLT1 and PLT2 transgenicrice plants grown under reduced nitrogen availability conditions, areshown in Table L, in absolute values (averaged events) and as apercentage compared to wild type plants. A substantial increase in TKWis observed for the transgenic seeds containing either construct,compared to wild type seeds.

TABLE L Results of TKW measurements of the T1 seeds of PLT1 and PLT2transgenic plants grown under reduced nitrogen availability conditions.TKW (g) % increase PLT1 transgenics 0.0295 12% PLT2 transgenics 0.02788% WT 0.0256

29.3 Results of the Phenotypic Evaluation of the Transgenics PlantsGrown Under Normal Growth Conditions, Expressing PLT2 Nucleic AcidSequence Under the Control of a Meristem-Specific Promoter

The TKW measurement results of the T1 seeds PLT2 transgenic rice plantsgrown under normal growth conditions, expressing PLT2 nucleic acidsequence under the control of a meristem-specific promoter, are shown inTable M, in absolute values (averaged events) and as a percentagecompared to wild type plants. A substantial increase in TKW is observedfor the transgenic seeds containing the construct, compared to wild typeseeds.

TABLE M Results of TKW measurements of the T1 seeds of PLT2 transgenicplants grown under normal growth conditions, expressing PLT2 nucleicacid sequence under the control of a mersitem-specific promoter. TKW (g)% increase PLT2 transgenics 0.0263 3% WT 0.0254

Example Section E bHLH Example 30 Identification of Paralogues of thebHLH of SEQ ID NO: 213 in Rice

SEQ ID 2 was used to search for paralogues in the rice genome using aBLASTP algorithm used to perform similarity searches of a protein querysequence to a given database of protein sequences. The protein databasequeried corresponded to the rice proteome of the MIPS institute, theMIPS Oryza sativa Database (MOsDB), comprising 59,712 sequences;27,051,637 total letters. Results were ranked according to highestsimilarity as determined from highest score and lowest e-value. Hitsidentifying bHLH protein sequences paralogous to SEQ ID NO: 2 had ascore of at least 50 and an e-value lower than e-05. Pairwise alignmentsbetween the query sequence and the de novo identified paralogues areshown below.

Query: SEQID 2 Smallest Sum High ProbabilitySequences producing High-scoring Segment Pairs: Score P(N) N9629.m00093|protein Helix-loop-helix DNA-binding domain, . . . 11321.3e−115   19629.m00090|protein Helix-loop-helix DNA-binding domain, . . .  3055.7e−28   19630.m01262|protein Helix-loop-helix DNA-binding domain, . . .  1891.1e−15   19629.m07159|protein Helix-loop-helix DNA-binding domain, . . .  1123.4e−05  1 >9629.m00093|protein Helix-loop-helix DNA-binding domain, putativeLength = 225 Score = 1132 (403.5 bits), Expect = 1.3e−115, P = 1.3e−115Identities = 224/224 (100%), Positives = 224/224 (100%) Query:   1MKSRKNSTTSTKAAGSCHTSSSGGGGGGGNCYSSSSSKMERKDVEKNRRLHMKGLCLKLS  60MKSRKNSTTSTKAAGSCHTSSSGGGGGGGNCYSSSSSKMERKDVEKNRRLHMKGLCLKLS Sbjct:   1MKSRKNSTTSTKAAGSCHTSSSGGGGGGGNCYSSSSSKMERKDVEKNRRLHMKGLCLKLS  60 Query: 61 SLIPAAAPRRHHHHYSTSSSSSPPSSTKEAVTQLDHLEQAAAYIKQLKGRIDELKKRKQQ 120SLIPAAAPRRHHHHYSTSSSSSPPSSTKEAVTQLDHLEQAAAYIKQLKGRIDELKKRKQQ Sbjct:  61SLIPAAAPRRHHHHYSTSSSSSPPSSTKEAVTQLDHLEQAAAYIKQLKGRIDELKKRKQQ 120 Query:121 AAALTTSTSNGGGGGMPVVEVRCQDGTLDVVVVSEAIREERERAVRLHEVIGVLEEEGAE 180AAALTTSTSNGGGGGMPVVEVRCQDGTLDVVVVSEAIREERERAVRLHEVIGVLEEEGAE Sbjct: 121AAALTTSTSNGGGGGMPVVEVRCQDGTLDVVVVSEAIREERERAVRLHEVIGVLEEEGAE 180 Query:181 VVNASFSVVGDKIFYTLHSQALCSRIGLDASRVSHRLRNLLLQY 224VVNASFSVVGDKIFYTLHSQALCSRIGLDASRVSHRLRNLLLQY Sbjct: 181VVNASFSVVGDKIFYTLHSQALCSRIGLDASRVSHRLRNLLLQY224 >9629.m00090|protein Helix-loop-helix DNA-binding domain, putativeLength = 363 Score = 305 (112.4 bits), Expect = 5.7e−28, P = 5.7e−28Identities = 87/230 (37%), Positives = 126/230 (54%) Query:  19TSSSGGGGGGGNCYSSSSSKMERKDVEKNRRLHMKGLCLKLSSLIPAAAPRRHHHHYSTS  78TSSSG G       +++++  ERK++E+ RR  MKGLC+KL+SLIP     + H   S Sbjct:  18TSSSGSGASS----TAAAAAAERKEMERRRRQDMKGLCVKLASLIP-----KEHCSMSKM  68 Query: 79 SSSSPPSSTKEAVTQLDHLEQAAAYIKQLKGRIDELKKRKQQ----------------AA 122 ++S         TQL  L++AAAYIK+LK R+DEL  ++                  AA Sbjct:  69QAASR--------TQLGSLDEAAAYIKKLKERVDELHHKRSMMSITSSRCRSGGGGGPAA 120 Query:123 ALTTSTSNGGGGGMPVVEVRCQDGTLDVVVVSEAIRE------------ERERAVRLHEV 170A   STS GGGG     ++        VV V + ++E               R V+ H+V Sbjct: 121AAGQSTSGGGGGEEEEEDMTRTTAAAAVVEVRQHVQEGSLISLDVVLICSAARPVKFHDV 180 Query:171 IGVLEEEGAEVVNASFSVVGDKIFYTLHSQALCSRIGLDASRVSHRLRNL 220I VLEEEGA++++A+FS+     +YT++S+A  SRIG++ASR+S RLR L Sbjct: 181ITVLEEEGADIISANFSLAAHNFYYTIYSRAFSSRIGIEASRISERLRAL230 >9630.m01262|protein Helix-loop-helix DNA-binding domain, putativeLength = 211 Score = 189 (71.6 bits), Expect = 1.1e−15, P = 1.1e−15Identities = 66/214 (30%), Positives = 102/214 (47%) Query:  21SSGGGGGGGNCYSSSSSKMERKDVEKNRRLHMKGLCLKLSSLIPAAAPRRHH-HHYSTSS  79S+GGGGGGG        K +RK  E+ RR  M  L   L SL+ +A P  +    +S S+ Sbjct:   7SAGGGGGGG--------KPDRKTTERIRREQMNKLYSHLDSLVRSAPPTVNSIPSHSNSN  58 Query: 80 SSSPPSSTK------EAVTQLDHLEQAAAYIKQLKGRIDELKKRKQQ---AAALTTSTSN 130S       +       A T+ D L  AA YI+Q + R+D L+++K++        +S+S+ Sbjct:  59SKYHQRKLRILGGAAAATTRPDRLGVAAEYIRQTQERVDMLREKKRELTGGGGGGSSSSS 118 Query:131 GGGGGM---PVVEVRCQDGTLDVVVVSEAIREERERAVRLHEVIGVLEEEGAEVVNASFS 187G G      P VEV+     L  ++ + A   +       H  +  +E+ G +V NA FS Sbjct: 119GAGAATAAAPEVEVQHLGSGLHAILFTGAPPTD---GASFHRAVRAVEDAGGQVQNAHFS 175 Query:188 VVGDKIFYTLHSQALCSRIGLDASRVSHRLRNLL 221 V G K  YT+H+       G++ RV  RL+  + Sbjct: 176 VAGAKAVYTIHAMIGDGYGGIE--RVVQRLKEAI 207

Example 31 Identification of Orthologues of the bHLH of SEQ ID NO: 213in Arabidopsis thaliana

SEQ ID 2 was used to search for orthologues in the Arabidopsis genomeusing a BLASTP algorithm used to perform a similarity search of aprotein query sequence to a given database of protein sequences. Theprotein database queried corresponded to the Arabidopsis proteome of theMIPS institute, the MIPS Arabidopsis thaliana database (MAtDB)comprising 26,735 sequences; 11,317,104 total letters. Results wereranked according to highest similarity as determined from highest scoreand lowest e-value. Hits identifying bHLH protein sequences orthologousto SEQ ID 2 had a score of at least 50 and an e-value lower than e-05.Pairwise alignments between the query sequence and the de novoidentified orthologue are shown below.

Query: SEQID 2 Database: /home/data/blast/orgs_sets/arabi26,735 sequences; 11,317,104 total letters. Smallest Sum HighProbability Sequences producing High-scoring Segment Pairs: Score P(N) NAt1g10585 unknown protein 180 4.5e−15   1 At4g20970 hypothetical protein149  8.7e−12   1 At4g25410 putative protein 118  2.0e−06   1At5g51780 putative bHLH transcription factor (bHLH036) 105 1.5e−05   1At5g51790 putative protein 110  1.5e−05   1 >At1g10585 unknown proteinLength = 122 Score = 180 (68.4 bits), Expect = 4.5e−15, P = 4.5e−15Identities = 38/119 (31%), Positives = 72/119 (60%) Query: 106QLKGRIDELKKRKQQAAALTTSTSNGGGGGMPVVEVRCQDGTLDVVVVSEAIREERERAV 165 QLK  ++LK++K+            G   +P + +R +D T+++ ++ + +  +R   V Sbjct:   3QLKENVNYLKEKKRTLLQGELGNLYEGSFLLPKLSIRSRDSTIEMNLIMD-LNMKR---V  58 Query:166 RLHEVIGVLEEEGAEVVNASFSVVGDKIFYTLHSQALCSRIGLDASRVSHRLRNLLLQY 224 LHE++ + EEEGA+V++A+   + D+  YT+ +QA+ SRIG+D SR+  R+R ++  Y Sbjct:  59MLHELVSIFEEEGAQVMSANLQNLNDRTTYTIIAQAIISRIGIDPSRIEERVRKIIYGY117 >At4g20970 hypothetical protein Length = 167 Score =149 (57.5 bits), Expect = 8.7e−12, P = 8.7e−12 Identities =49/171 (28), Positives = 86/171 (50%) Query:  33 SSSSSKMERKDVEKNRRLHMKGLCLKLSSLIPAAAPRRHHHHYSTSSSSSPPSSTKEAVT  92 + S  ++RK VEKNRR+ MK L  +L SL+P        HH ST   + P     EA Sbjct:   8 TGQSRSVDRKTVEKNRRMQMKSLYSELISLLP--------HHSSTEPLTLP-DQLDEAAN  58 Query: 93  QLDHLEQAAAYIKQLKGRI---DELKKRKQQ-AAALTTSTSNGGGGGMPVVEVRCQDGTL 148  + L+      ++ K  +     L+K     ++++++S        +P +E++ + G++ Sbjct:  59 YIKKLQVNVEKKRERKRNLVATTTLEKLNSVGSSSVSSSVDVSVPRKLPKIEIQ-ETGSI 117 Query:149 DVVVVSEAIREERERAVRLHEVIGVLEEE-GAEVVNASFSVVGDKIFYTLH 198   + +  ++   E      E+I VL EE GAE+ +A +S+V D +F+TLH Sbjct: 118FHIFLVTSL----EHKFMFCEIIRVLTEELGAEITHAGYSIVDDAVFHTLH 164

Example 32 Identification of a bHLH from Medicago truncatula

The question to be addressed was whether the query sequence (SEQ ID NO:227 from Medicago truncatula) was a bHLH polypeptide according to thedefinition applied herein. SEQ ID 227 was compared to the Arabidopsisproteome database of the MIPS institute.

Comparison was carried out using the BLASTP 2.0 MP-WashU algorithm,which performs similarity searches of a protein query sequence to agiven database of protein sequences. (Reference: Gish, W. (1996-2002)).The parameters used in the comparison were E value 10 Cutoff score (S2):56

The protein database queried corresponded to the Arabidopsis proteome ofthe MIPS institute comprising 26,735 sequences (Database:/home/data/blast/orgs_sets/arabi 26,735 sequences; 11,317,104 totalletters). Results were ranked according to highest similarity asdetermined from highest score and lowest e-value. The first Hitidentified (underlined in the alignment) corresponded to SEQ ID NO: 225(At4g20970) indicating that the sequence is a bHLH polypeptide accordingto the definition applied herein. Pairwise alignments between the querysequence from Medicago and the first hit corresponding to the de novoidentified bHLH are shown below.

BLASTP 2.0 MP-WashU [9 Sep. 2002] [decunix4.0-ev56-I32LPF642002-09-09T17:45:09]

Query = SEQ ID NO: 227 (140 letters)Database: /home/data/blast/orgs_sets/arabi26,735 sequences; 11,317,104 total letters.Searching....10....20....30....40....50....60....70....80....90....100% doneSmallest Sum High ProbabilitySequences producing High-scoring Segment Pairs: Score P(N) NAt4g20970 hypothetical protein 248 2.8e−22   1At5g51780 putative bHLH transcription factor (bHLH036) 103  6.5e−07   1At1g10585 unknown protein 100  1.4e−06   1At4g25400 putative bHLH transcription factor (bHLH118)  99 7.3e−06  1 >At4g20970 unknown protein Length = 167 Score =248 (92.4 bits), Expect = 2.8e−22, P = 2.8e−22 Identities =52/123 (42%), Positives = 82/123 (66%) Query:   7EAISVPDQLKEATNYIKKLQINLEKMKEKKNFLLG---IQRPN------VNLNRNQKMGL  57E +++PDQL EA NYIKKLQ+N+EK +E+K  L+    +++ N      V+ + +  + Sbjct:  45EPLTLPDQLDEAANYIKKLQVNVEKKRERKRNLVATTTLEKLNSVGSSSVSSSVDVSVPR 104 Query: 58 KSPKIKIQQIGLVLEVVLITGLESQFLFSETFRVLHEE-GVDIVNASYKVNEDSVFHSIH 116K PKI+IQ+ G +  + L+T LE +F+F E  RVL EE G +I +A Y + +D+VFH++H Sbjct: 105KLPKIEIQETGSIFHIFLVTSLEHKFMFCEIIRVLTEELGAEITHAGYSIVDDAVFHTLH 164 Query:117 CQV 119 C+V Sbjct: 165 CKV 166

Example 33 Identification of Sequences Related to the Nucleic AcidSequence Used in the Methods of the Invention

bHLH Sequences, whether nucleotide (full length cDNA, ESTs or genomic)or protein (full length or partial polypeptides), were used toidentified other bHLH nucleotide or proteins sequences amongst thosemaintained in the Entrez Nucleotides database at the National Center forBiotechnology Information (NCBI) using database sequence search tools,such as the Basic Local Alignment Tool (BLAST) (Altschul et al. (1990)J. Mol. Biol. 215:403-410; and Altschul et al. (1997) Nucleic Acids Res.25:3389-3402). The program was used to find regions of local similaritybetween sequences by comparing nucleic acid or polypeptide sequences tosequence databases and by calculating the statistical significance ofmatches. For example, the polypeptide encoded by SEQ ID NO: 213 was usedas query to the nr (non-redundant: (Database: All GenBank+EMBL+DDBJ+PDBsequences (but no EST, STS, GSS, environmental samples or phase 0, 1 or2 HTGS sequences) 3,819,973 sequences; 16,928,533,343 total letters)database sequence using the TBLASTN algorithm, with default settings andthe filter to ignore low complexity sequences. The output of theanalysis (shown below) was viewed by pairwise comparison, and rankedaccording to the probability score (E-value), where the score reflectthe probability that a particular alignment occurs by chance (the lowerthe E-value, the more significant the hit). In addition to E-values,comparisons were also scored by percentage identity. Percentage identityrefers to the number of identical nucleotides (or amino acids) betweenthe two compared nucleic acid (or polypeptide) sequences over aparticular length.

Hits on the database identifying BHLH proteins produced a score of atleast 50 and an e-value equal to or lower than e-05.

Score E Sequences producing significant alignments: (Bits)  Valuegi|55765745|ref|NM_183508.2| Oryza sativa (japonica cultivar-gro 4401e−121 gi|42821746|dbj|AK109432.2|Oryza sativa (japonica cultivar-g . . . 440 1e−121gi|55769744|ref|XM_549862.1| Oryza sativa (japonica cultivar-gro 2471e−105 gi|58530787|dbj|AP008207.1|Oryza sativa (japonica cultivar-g . . . 263 2e−68gi|17385651|dbj|AP002845.3| Oryza sativa (japonica cultivar-g . . . 2632e−68 gi|32980356|dbj|AK070332.1|Oryza sativa (japonica cultivar-g . . . 251 9e−65gi|34894135|ref|NM_183504.1| Oryza sativa (japonica cultivar-gro 1373e−30 gi|15293050|gb|AY050959.1|Arabidopsis thaliana unknown prote . . .  80.1 6e−16gi|19340923|ref|NM_100934.2| Arabidopsis thaliana transcripti . . . 87.8 2e−15 gi|58531195|dbj|AP008214.1|Oryza sativa (japonica cultivar-g . . .  51.6 4e−13gi|42408246|dbj|AP004557.3| Oryza sativa (japonica cultivar-g . . . 51.6 4e−13 gi|42408168|dbj|AP004376.3|Oryza sativa (japonica cultivar-g . . .  51.6 4e−13gi|22328837|ref|NM_118215.2| Arabidopsis thaliana DNA binding . . . 78.2 1e−12gi|7268888|emb|AL161554.2|ATCHRIV54 Arabidopsis thaliana DNA chr  77.82e−12gi|5262774|emb|AL080282.1|ATT13K14 Arabidopsis thaliana DNA c . . . 77.8 2e−12 gi|50906596|ref|XM_464787.1|Oryza sativa (japonica cultivar-gro  77.0 3e−12

Example 34 Determination of Global Similarity and Identity Between bHLHTranscription Factors

Global percentages similarity and identity between bHLH polypeptides wasdetermined using the MatGAT software (BMC Bioinformatics. 2003 Jul. 10;4:29. MatGAT: an application that generates similarity/identity matricesusing protein or DNA sequences. Campanella J J, Bitincka L, Smalley J.).MatGAT software generates similarity/identity matrices for DNA orprotein sequences without needing pre-alignment of the data. The programperforms a series of pair-wise alignments, calculates similarity andidentity, and then places the results in a distance matrix.

Parameters used in the comparison were:

-   -   Scoring matrix: Blosum62    -   First Gap: 12    -   Extending gap: 2

Results are shown in FIG. 19 a.

Example 35 Determination of Global Similarity and Identity Between bHLHDomains in bHLH Polypeptides

bHLH domains were mapped using the SMART software (Schultz et al. (1998)Proc. Natl. Acad. Sci. USA 95, 5857-5864; Letunic et al. (2006) NucleicAcids Res 34, D257-D260). Smart software is available through EMBLinstitute (European Molecular Biology Laboratory (EMBL).

The sequences of the bHLH domain used are given below.

Hv_BHLH KESEKERRKRMKALCEKLASLIPREHCCSTTDTMTQLGSLDVGASYIKKLKERVDEOS_NP_908393_BHLHKEMERRRRQDMKGLCVKLASLIPKEHCSMSKMQAASRTQLGSLDEAAAYIKKLKERVDEOS_NP_908397.1_BHLHKDVEKNRRLHMKGLCLKLSSLIPAAAPRRHHHHYSTSSSSSPPSSTKEAVTQLDHLEQAAAYIKQLKGRIDE Os_XP_464787.1_BHLHKTTERIRREQMNKLYSHLDSLVRSAPPTVNSIPSHSNSNSKYHQRKLRILGGAAAATTRPDRLGVAAEYIRQTQERVDM AT1G10585_bHLHnlrekdrrmrmkhlfsilsshvsptrklpvphlidqatsymiqlkenvny At4g20970_bHLHKTVEKNRRMQMKSLYSELISLLPHHSSTEPLTLPDQLDEAANYIKKLQVNVEK

Global percentages of similarity and identity between bHLH domains ofbHLH polypeptides were determined using the software and parametersdescribed in Example 34.

Results are shown in FIG. 19 b.

Example 36 Cloning of the Nucleic Acid Sequence Used in the Methods ofthe Invention

The nucleic acid sequence used in the methods of the invention wasamplified by PCR using as template a custom-made Oryza sativa seedlingscDNA library (in pCMV Sport 6.0; Invitrogen, Paisley, UK). PCR wasperformed using Hifi Taq DNA polymerase in standard conditions, using200 ng of template in a 50 μl PCR mix. The primers used were prm06808(SEQ ID NO: 231; sense, start codon in bold, AttB1 site in italic:5′-ggggacaagtttgtacaaaaaagcaggcttaaacaatgaagagcaggaagaacagc 3′) andprm06809 (SEQ ID NO: 232; reverse, complementary, AttB2 site in italic:5′ ggggaccactttgtacaagaaagctgggtgcagagtgaaagagtggtgtg 3′), which includethe AttB sites for Gateway recombination. The amplified PCR fragment waspurified also using standard methods. The first step of the Gatewayprocedure, the BP reaction, was then performed, during which the PCRfragment recombines in vivo with the pDONR201 plasmid to produce,according to the Gateway terminology, an “entry clone”, pbHLH. PlasmidpDONR201 was purchased from Invitrogen, as part of the Gateway®technology.

Example 37 Expression Vector Construction

The entry clone p076 was subsequently used in an LR reaction with pGOS2,a destination vector used for Oryza sativa transformation. This vectorcontains as functional elements within the T-DNA borders: a plantselectable marker; a screenable marker expression cassette; and aGateway cassette intended for LR in vivo recombination with the nucleicacid sequence of interest already cloned in the entry clone. A rice GOS2promoter (SEQ ID NO: 233, alternatively, SEQ ID NO: 230 is equallyuseful) for constitutive expression (internal reference PRO0129) waslocated upstream of this Gateway cassette.

After the LR recombination step, the resulting expression vectorpGOS2::bHLH (FIG. 20) was transformed into Agrobacterium strain LBA4044and subsequently to Oryza sativa plants.

Example 38 Plant Transformation Rice Transformation

The Agrobacterium containing the expression vector was used to transformOryza sativa plants. Mature dry seeds of the rice japonica cultivarNipponbare were dehusked. Sterilization was carried out by incubatingfor one minute in 70% ethanol, followed by 30 minutes in 0.2% HgCl₂,followed by a 6 times 15 minutes wash with sterile distilled water. Thesterile seeds were then germinated on a medium containing 2,4-D (callusinduction medium). After incubation in the dark for four weeks,embryogenic, scutellum-derived calli were excised and propagated on thesame medium. After two weeks, the calli were multiplied or propagated bysubculture on the same medium for another 2 weeks. Embryogenic calluspieces were sub-cultured on fresh medium 3 days before co-cultivation(to boost cell division activity).

Agrobacterium strain LBA4404 containing the expression vector was usedfor co-cultivation. Agrobacterium was inoculated on AB medium with theappropriate antibiotics and cultured for 3 days at 28° C. The bacteriawere then collected and suspended in liquid co-cultivation medium to adensity (OD₆₀₀) of about 1. The suspension was then transferred to aPetri dish and the calli immersed in the suspension for 15 minutes. Thecallus tissues were then blotted dry on a filter paper and transferredto solidified, co-cultivation medium and incubated for 3 days in thedark at 25° C. Co-cultivated calli were grown on 2,4-D-containing mediumfor 4 weeks in the dark at 28° C. in the presence of a selection agent.During this period, rapidly growing resistant callus islands developed.After transfer of this material to a regeneration medium and incubationin the light, the embryogenic potential was released and shootsdeveloped in the next four to five weeks. Shoots were excised from thecalli and incubated for 2 to 3 weeks on an auxin-containing medium fromwhich they were transferred to soil. Hardened shoots were grown underhigh humidity and short days in a greenhouse.

Approximately 30 independent T0 rice transformants were generated forone construct. The primary transformants were transferred from a tissueculture chamber to a greenhouse. After a quantitative PCR analysis toverify copy number of the T-DNA insert, only single copy transgenicplants that exhibit tolerance to the selection agent were kept forharvest of T1 seed. Seeds were then harvested three to five months aftertransplanting. The method yielded single locus transformants at a rateof over 50% (Aldemita and Hodges 1996, Chan et al. 1993, Hiei et al.1994).

Corn Transformation

Transformation of maize (Zea mays) is performed with a modification ofthe method described by Ishida et al. (1996) Nature Biotech 14(6):745-50. Transformation is genotype-dependent in corn and only specificgenotypes are amenable to transformation and regeneration. The inbredline A188 (University of Minnesota) or hybrids with A188 as a parent aregood sources of donor material for transformation, but other genotypescan be used successfully as well. Ears are harvested from corn plantapproximately 11 days after pollination (DAP) when the length of theimmature embryo is about 1 to 1.2 mm. Immature embryos are cocultivatedwith Agrobacterium tumefaciens containing the expression vector, andtransgenic plants are recovered through organogenesis. Excised embryosare grown on callus induction medium, then maize regeneration medium,containing the selection agent (for example imidazolinone but variousselection markers can be used). The Petri plates are incubated in thelight at 25° C. for 2-3 weeks, or until shoots develop. The green shootsare transferred from each embryo to maize rooting medium and incubatedat 25° C. for 2-3 weeks, until roots develop. The rooted shoots aretransplanted to soil in the greenhouse. T1 seeds are produced fromplants that exhibit tolerance to the selection agent and that contain asingle copy of the T-DNA insert.

Wheat Transformation

Transformation of wheat is performed with the method described by Ishidaet al. (1996) Nature Biotech 14(6): 745-50. The cultivar Bobwhite(available from CIMMYT, Mexico) is commonly used in transformation.Immature embryos are co-cultivated with Agrobacterium tumefacienscontaining the expression vector, and transgenic plants are recoveredthrough organogenesis. After incubation with Agrobacterium, the embryosare grown in vitro on callus induction medium, then regeneration medium,containing the selection agent (for example imidazolinone but variousselection markers can be used). The Petri plates are incubated in thelight at 25° C. for 2-3 weeks, or until shoots develop. The green shootsare transferred from each embryo to rooting medium and incubated at 25°C. for 2-3 weeks, until roots develop. The rooted shoots aretransplanted to soil in the greenhouse. T1 seeds are produced fromplants that exhibit tolerance to the selection agent and that contain asingle copy of the T-DNA insert.

Soybean Transformation

Soybean is transformed according to a modification of the methoddescribed in the Texas A&M U.S. Pat. No. 5,164,310. Several commercialsoybean varieties are amenable to transformation by this method. Thecultivar Jack (available from the Illinois Seed foundation) is commonlyused for transformation. Soybean seeds are sterilised for in vitrosowing. The hypocotyl, the radicle and one cotyledon are excised fromseven-day old young seedlings. The epicotyl and the remaining cotyledonare further grown to develop axillary nodes. These axillary nodes areexcised and incubated with Agrobacterium tumefaciens containing theexpression vector. After the cocultivation treatment, the explants arewashed and transferred to selection media. Regenerated shoots areexcised and placed on a shoot elongation medium. Shoots no longer than 1cm are placed on rooting medium until roots develop. The rooted shootsare transplanted to soil in the greenhouse. T1 seeds are produced fromplants that exhibit tolerance to the selection agent and that contain asingle copy of the T-DNA insert.

Rapeseed/Canola Transformation

Cotyledonary petioles and hypocotyls of 5-6 day old young seedling areused as explants for tissue culture and transformed according to Babicet al. (1998, Plant Cell Rep 17: 183-188). The commercial cultivarWestar (Agriculture Canada) is the standard variety used fortransformation, but other varieties can also be used. Canola seeds aresurface-sterilized for in vitro sowing. The cotyledon petiole explantswith the cotyledon attached are excised from the in vitro seedlings, andinoculated with Agrobacterium (containing the expression vector) bydipping the cut end of the petiole explant into the bacterialsuspension. The explants are then cultured for 2 days on MSBAP-3 mediumcontaining 3 mg/l BAP, 3% sucrose, 0.7% Phytagar at 23° C., 16 hr light.After two days of co-cultivation with Agrobacterium, the petioleexplants are transferred to MSBAP-3 medium containing 3 mg/l BAP,cefotaxime, carbenicillin, or timentin (300 mg/l) for 7 days, and thencultured on MSBAP-3 medium with cefotaxime, carbenicillin, or timentinand selection agent until shoot regeneration. When the shoots are 5-10mm in length, they are cut and transferred to shoot elongation medium(MSBAP-0.5, containing 0.5 mg/l BAP). Shoots of about 2 cm in length aretransferred to the rooting medium (MS0) for root induction. The rootedshoots are transplanted to soil in the greenhouse. T1 seeds are producedfrom plants that exhibit tolerance to the selection agent and thatcontain a single copy of the T-DNA insert.

Alfalfa Transformation

A regenerating clone of alfalfa (Medicago sativa) is transformed usingthe method of (McKersie et al., 1999 Plant Physiol 119: 839-847).Regeneration and transformation of alfalfa is genotype dependent andtherefore a regenerating plant is required. Methods to obtainregenerating plants have been described. For example, these can beselected from the cultivar Rangelander (Agriculture Canada) or any othercommercial alfalfa variety as described by Brown D C W and A Atanassov(1985. Plant Cell Tissue Organ Culture 4: 111-112). Alternatively, theRA3 variety (University of Wisconsin) has been selected for use intissue culture (Walker et al., 1978 Am J Bot 65:654-659). Petioleexplants are cocultivated with an overnight culture of Agrobacteriumtumefaciens C58C1 pMP90 (McKersie et al., 1999 Plant Physiol 119:839-847) or LBA4404 containing the expression vector. The explants arecocultivated for 3 d in the dark on SH induction medium containing 288mg/L Pro, 53 mg/L thioproline, 4.35 g/L K2SO4, and 100 μmacetosyringinone. The explants are washed in half-strengthMurashige-Skoog medium (Murashige and Skoog, 1962) and plated on thesame SH induction medium without acetosyringinone but with a suitableselection agent and suitable antibiotic to inhibit Agrobacterium growth.After several weeks, somatic embryos are transferred to BOi2Ydevelopment medium containing no growth regulators, no antibiotics, and50 g/L sucrose. Somatic embryos are subsequently germinated onhalf-strength Murashige-Skoog medium. Rooted seedlings were transplantedinto pots and grown in a greenhouse. T1 seeds are produced from plantsthat exhibit tolerance to the selection agent and that contain a singlecopy of the T-DNA insert.

Example 39 Evaluation Procedure 39.1 Evaluation Setup

Approximately 30 independent T0 rice transformants were generated. Theprimary transformants were transferred from a tissue culture chamber toa greenhouse for growing and harvest of T1 seed. Seven events, of whichthe T1 progeny segregated 3:1 for presence/absence of the transgene,were retained. For each of these events, approximately 10 T1 seedlingscontaining the transgene (hetero- and homo-zygotes) and approximately 10T1 seedlings lacking the transgene (nullizygotes) were selected bymonitoring visual marker expression. The transgenic plants and thecorresponding nullizygotes were grown side-by-side at random positions.Greenhouse conditions were of shorts days (12 hours light), 28° C. inthe light and 22° C. in the dark, and a relative humidity of 70%.

Four T1 events were further evaluated in the T2 generation following thesame evaluation procedure as for the T1 generation but with moreindividuals per event. From the stage of sowing until the stage ofmaturity the plants were passed several times through a digital imagingcabinet. At each time point digital images (2048×1536 pixels, 16 millioncolours) were taken of each plant from at least 6 different angles.

39.2 Statistical Analysis: t-Test and F-Test

A two factor ANOVA (analysis of variants) was used as a statisticalmodel for the overall evaluation of plant phenotypic characteristics. AnF-test was carried out on all the parameters measured of all the plantsof all the events transformed with the gene of the present invention.The F-test was carried out to check for an effect of the gene over allthe transformation events and to verify for an overall effect of thegene, also known as a global gene effect. The threshold for significancefor a true global gene effect was set at a 5% probability level for theF-test. A significant F-test value points to a gene effect, meaning thatit is not only the mere presence or position of the gene that is causingthe differences in phenotype.

To check for an effect of the genes within an event, i.e., for aline-specific effect, a t-test was performed within each event usingdata sets from the transgenic plants and the corresponding null plants.“Null plants” or “null segregants” or “nullizygotes” are the plantstreated in the same way as the transgenic plant, but from which thetransgene has segregated. Null plants can also be described as thehomozygous negative transformed plants. The threshold for significancefor the t-test is set at 10% probability level. The results for someevents can be above or below this threshold. This is based on thehypothesis that a gene might only have an effect in certain positions inthe genome, and that the occurrence of this position-dependent effect isnot uncommon. This kind of gene effect is also named herein a “lineeffect of the gene”. The p-value is obtained by comparing the t-value tothe t-distribution or alternatively, by comparing the F-value to theF-distribution. The p-value then gives the probability that the nullhypothesis (i.e., that there is no effect of the transgene) is correct.

Example 40 Evaluation Results

Early vigour was determined by counting the total number of pixels fromaboveground plant parts discriminated from the background. This valuewas averaged for the pictures taken on the same time point fromdifferent angles and was converted to a physical surface value expressedin square mm by calibration. The results described below are for plantsthree weeks post-germination.

Early vigour (as determined by aboveground area) was seen in six out ofseven events of the T1 generation, with an overall increase inaboveground area for transgenic seedlings of 22% compared to controlplants. Four of these T1 events were further evaluated in the T2generation, and all four of these events gave an increase in abovegroundarea for transgenic seedlings compared to control plants, with anoverall increase in aboveground area for transgenic seedlings of 13%compared to control plants. The results were also shown to bestatistically significant with the p-value from the F-test being 0.0007(T2 generation) indicating that the effect seen is likely due to thetransgene rather than the position of the gene or a line effect.

Example Section F SPL15 Example 41 Identification of Sequences Relatedto the Nucleic Acid Sequence Used in the Methods of the Invention

Sequences (full length cDNA, ESTs or genomic) related to the nucleicacid sequence used in the methods of the present invention wereidentified amongst those maintained in the Entrez Nucleotides databaseat the National Center for Biotechnology Information using databasesequence search tools, such as the Basic Local Alignment Tool (BLAST)(Altschul et al. (1990) J. Mol. Biol. 215:403-410; and Altschul et al.(1997) Nucleic Acids Res. 25:3389-3402). The program is used to findregions of local similarity between sequences by comparing nucleic acidor polypeptide sequences to sequence databases and by calculating thestatistical significance of matches. The polypeptide encoded by thenucleic acid of the present invention was used for the TBLASTNalgorithm, with default settings and the filter to ignore low complexitysequences set off. The output of the analysis was viewed by pairwisecomparison, and ranked according to the probability score (E-value),where the score reflect the probability that a particular alignmentoccurs by chance (the lower the E-value, the more significant the hit).In addition to E-values, comparisons were also scored by percentageidentity. Percentage identity refers to the number of identicalnucleotides (or amino acids) between the two compared nucleic acid (orpolypeptide) sequences over a particular length. In some instances, thedefault parameters may be adjusted to modify the stringency of thesearch

The Table N below provides a list of nucleic acid sequences related tothe nucleic acid sequence used in the methods of the present invention.

TABLE N nucleic acid sequences related to the nucleic acid sequence (SEQID NO: 234) used in the methods of the present invention, and thecorresponding deduced polypeptides Nucleic acid Polypeptide SequenceNCBI accession Name SEQ ID NO SEQ ID NO length number Source organismArath_SPL15 234 235 Full length NM_115654.1 Arabidopsis thaliana(At3g57920) Arath_SPL9 236 237 Full length AY150378 Arabidopsis thaliana(At2g42200) Aqufo_SPL 238 239 Full length contig of DR915312 Aquilegiaformosa × DR949057.1 Aquilegia pubescens Goshi_SPL 240 241 Full lengthDT566400 Gossypium hirsutum Iponi_SPL 242 243 Full length contig ofIpomoea nil BJ576204.1 BJ556115 BJ567301 Lacsa_SPL 244 245 Full lengthcontig of DY966949 Lactuca sativa DW119178 Maldo_SPL 246 247 Full lengthcontig of Malus domestica CN891102.1 CO868185.1 CV523507 Medtr_SPL 248249 Full length spliced from Medicago truncatula AC170989.2 Nicbe_SPL250 251 Full length contig of CK284078.1 Nicotiana CK294165 bentamianaOrysa_SPL 252 253 Full length XM_483285 Oryza sativa Orysa_SPL II 254255 Full length spliced from AC108762 Oryza sativa Soltu_SPL 256 257Full length contig of CK246692.1 Solanum tuberosum CK254420.1 Vitvi_SPL258 259 Full length contig of Vitis vinifera CV098277 CV092812.1Zeama_SPL 260 261 Full length contig of EB160653 Zea mays DY235599DV029129 Zeama_SPL 262 263 Full length contig of AJ011619 Zea mays IIDV033513.1 DY532686.1 Sorpr_SPL 264 265 Partial BF422188 Sorghumpropinquium Allce_SPL 266 267 Partial CF444518.1 Allium cepa Antma_SPL268 269 Partial AMA011623 Antirrhinum majus Brana_SPL 270 271 PartialCX189447 Brassica napus Sacof_SPL 272 273 Partial contig of CA113070Saccharum CA254724 officinarum Fesar_SPL 274 275 Partial DT706587.1Festuca arundinacea Brara_SPL 282 283 Full length AC189445.1 Brassicarapa Glyma_SPL 284 285 Full length CX708501.1 Glycine max BG651519.1Poptr_SPL 286 287 Full length scaff_XVI.416 Populus tremuloidesCitcl_SPL 288 289 Partial DY293795 Citrus clementina Betvu_SPL 290 291Partial BQ594361.1 Beta vulgaris Hevbr_SPL 292 293 Partial EC604947Hevea brasiliensis

Example 42 Determination of Global Similarity and Identity Between SPL15Transcription Factors, and their SPL DBD

Global percentages of similarity and identity between SPL15transcription factors were determined using one of the methods availablein the art, the MatGAT (Matrix Global Alignment Tool) software (BMCBioinformatics. 2003 4:29. MatGAT: an application that generatessimilarity/identity matrices using protein or DNA sequences. CampanellaJ J, Bitincka L, Smalley J; software hosted by Ledion Bitincka). MatGATsoftware generates similarity/identity matrices for DNA or proteinsequences without needing pre-alignment of the data. The programperforms a series of pair-wise alignments using the Myers and Millerglobal alignment algorithm (with a gap opening penalty of 12, and a gapextension penalty of 2), calculates similarity and identity using forexample Blosum 62 (for polypeptides), and then places the results in adistance matrix. Sequence similarity is shown in the bottom half of thedividing line and sequence identity is shown in the top half of thediagonal dividing line. The sequence of SEQ ID NO: 235 is indicated asnumber 5 in the matrix.

Parameters used in the comparison were:

-   -   Scoring matrix: Blosum62    -   First Gap: 12    -   Extending gap: 2

Results of the software analysis are shown in Table 0 for the globalsimilarity and identity over the full length of the SPL15 transcriptionfactor polypeptides. Percentage identity is given above the diagonal andpercentage similarity is given below the diagonal. Percentage identitybetween the SPL15 transcription factor paralogues and orthologues rangesbetween 30 and 70%, reflecting the relatively low sequence identityconservation between them outside of the SPL DBD.

TABLE O MatGAT results for global similarity and identity over the fulllength of the SPL15 transcription factor polypeptides. Global similarityand identity over the full length of the SPL15 transcription factorpolypeptides 1 2 3 4 5 6 7 8 9 10 11 12 13 14 1. Arath_SPL9 51.1 44.840.3 38.4 44 43.1 40.3 34.6 35.9 39.4 46.7 33.9 35.7 2. Arath_SPL15 63.239.4 39 33.7 40.1 40.3 35.1 31.3 33.7 36.5 39.6 30.5 31.9 3. Goshi_SPL58.9 53.2 47.3 43 54.4 46.9 52 41.3 40.2 50.1 59.8 39.5 39.1 4.Iponi_SPL 53.3 55.9 61.7 43.9 50.9 46.8 56.1 33.6 37.2 56.7 53.5 36.338.7 5. Lacsa_SPL 52 49.4 53.2 61.3 45.8 43.5 46.4 37 38.3 47.9 48.137.6 39.9 6. Maldo_SPL 59.3 54.5 64.3 63.5 56.3 49.5 57.4 42 38.1 55.268.7 38.9 40.6 7. Medtr_SPL 55.2 57.6 60.4 64.5 58.4 65.1 46.7 37.3 39.246.3 55.4 37.6 39.7 8. Nicbe_SPL 58.2 51.6 63.8 66.8 56.8 70.8 62.9 36.940 66.6 62.1 38.2 37.5 9. Orysa_SPL 48.7 42.2 54.2 47 47.2 53.2 49.251.8 62.4 37.3 40.7 59.5 54.9 10. Orysa_SPL II 49.9 45.8 53.4 49.1 48.951.1 53.4 53.2 72.2 37.6 43.6 54.3 62.9 11. Soltu_SPL 57.3 54 64 68.559.4 69.6 62.9 77.6 51.8 50.4 60.7 36.9 37.9 12. Vitvi_SPL 62.8 54.969.2 66 59.4 80.2 67.5 73.4 53.5 56.5 73.6 42.2 40.8 13. Zeama_SPL 47.343.3 55.2 48.8 49.3 52 51.2 52.5 68.3 67.9 49.8 54 54.9 14. Zeama_SPL II49.2 43.1 53.2 51.6 50.8 54.5 53.2 48.7 64.3 71.2 51.1 54.6 66.2

Results of the software analysis are shown in Table P for the globalsimilarity and identity over the SPL DBD of the SPL15 transcriptionfactor polypeptides. Percentage identity is given above the diagonal andpercentage similarity is given below the diagonal. Percentage identitybetween the SPL DBD of SPL15 transcription factor paralogues andorthologues ranges between 70% and 100%.

TABLE P MatGAT results for global similarity and identity over the SPLDBD of the SPL15 transcription factor polypeptides. Global similarityand identity over the SPL DBD of the SPL15 transcription factorpolypeptides 1 2 3 4 5 6 7 8 9 10 11 12 13 14 1. SPL\DBD\Aqufo_SPL 75.679.5 84.8 85.9 87.2 85.9 88.5 84.6 80.8 78.2 85.9 91 76.9 2.SPL\DBD\Arath_SPL15 82.1 78.2 83.5 82.1 78.2 76.9 78.2 79.5 71.8 74.479.5 79.5 71.8 3. SPL\DBD\Arath_SPL9 87.2 85.9 77.2 78.2 82.1 82.1 79.578.2 76.9 78.2 79.5 80.8 76.9 4. SPL\DBD\Goshi_SPL 88.6 91.1 86.1 86.184.8 84.8 84.8 84.8 78.5 78.5 86.1 89.9 78.5 5. SPL\DBD\Iponi_SPL 89.785.9 87.2 91.1 84.6 84.6 85.9 85.9 80.8 79.5 87.2 89.7 78.2 6.SPL\DBD\Lacsa_SPL 89.7 85.9 88.5 91.1 91 88.5 84.6 85.9 80.8 79.5 85.989.7 78.2 7. SPL\DBD\Maldo_SPL 92.3 82.1 88.5 89.9 91 91 84.6 89.7 85.983.3 88.5 91 80.8 8. SPL\DBD\Medtr_SPL 89.7 85.9 89.7 92.4 92.3 91 93.684.6 79.5 78.2 88.5 93.6 78.2 9. SPL\DBD\Nicbe_SPL 87.2 83.3 85.9 88.692.3 89.7 91 92.3 84.6 82.1 88.5 89.7 80.8 10. SPL\DBD\Orysa_SPL 87.280.8 84.6 84.8 88.5 85.9 89.7 85.9 89.7 85.9 84.6 83.3 83.3 11.SPL\DBD\Orysa_SPL II 84.6 82.1 87.2 83.5 85.9 84.6 88.5 85.9 88.5 92.382.1 82.1 84.6 12. SPL\DBD\Soltu_SPL 89.7 87.2 87.2 91.1 94.9 89.7 93.693.6 92.3 87.2 84.6 93.6 80.8 13. SPL\DBD\Vitvi_SPL 92.3 87.2 89.7 93.794.9 92.3 96.2 97.4 92.3 87.2 87.2 96.2 82.1 14. SPL\DBD\Zeama_SPL II83.3 79.5 84.6 83.5 84.6 83.3 84.6 87.2 84.6 88.5 89.7 83.3 85.9

Example 43 Cloning of the Nucleic Acid Sequence Used in the Methods ofthe Invention

DNA manipulation: unless otherwise stated, recombinant DNA techniquesare performed according to standard protocols described in (Sambrook(2001) Molecular Cloning: a laboratory manual, 3rd Edition Cold SpringHarbor Laboratory Press, CSH, New York) or in Volumes 1 and 2 of Ausubelet al. (1994), Current Protocols in Molecular Biology, CurrentProtocols. Standard materials and methods for plant molecular work aredescribed in Plant Molecular Biology Labfase (1993) by R. D. D. Croy,published by BIOS Scientific Publications Ltd (UK) and BlackwellScientific Publications (UK).

The nucleic acid sequence used in the methods of the invention wasamplified by PCR using as template a custom-made Arabidopsis thalianamixed tissues cDNA library (in pCMV Sport 6.0; Invitrogen, Paisley, UK).PCR was performed using Hifi Taq DNA polymerase in standard conditions,using 200 ng of template in a 50 μl PCR mix. The primers used wereprm07277 (SEQ ID NO: 280; sense, start codon in bold, AttB1 site inlower case: 5′-ggggacaagtttgtacaaaaaagcaggcttaaacaATGGAGTTGTTAATGTGTTCG3′) and prm07278 (SEQ ID NO: 281; reverse, complementary, AttB2 site inlower case: 5′ ggggaccactttgtacaagaaagctgggtTGATGAAGATCTTAAAAGGTGA 3′),which include the AttB sites for Gateway recombination. The amplifiedPCR fragment was purified also using standard methods. The first step ofthe Gateway procedure, the BP reaction, was then performed, during whichthe PCR fragment recombines in vivo with the pDONR201 plasmid toproduce, according to the Gateway terminology, an “entry clone”, pSPL15.Plasmid pDONR201 was purchased from Invitrogen, as part of the Gateway®technology.

Example 44 Expression Vector Construction

The entry clone p13075 was subsequently used in an LR reaction withp06659, a destination vector used for Oryza sativa transformation. Thisvector contains as functional elements within the T-DNA borders: a plantselectable marker; a screenable marker expression cassette; and aGateway cassette intended for LR in vivo recombination with the nucleicacid sequence of interest already cloned in the entry clone. A rice HMGBpromoter (SEQ ID NO: 294) for constitutive expression was locatedupstream of this Gateway cassette. Alternatively, the HMGB promoterrepresented by SEQ ID NO: 46 is equally useful. A similar construct wasmade with the SPL15 coding sequence under control of the constitutiveGOS2 promoter (SEQ ID NO: 295).

After the LR recombination step, the resulting expression vectorpHMGB::SPL15 (FIG. 26), or pGOS2::SPL15, was transformed intoAgrobacterium strain LBA4044 and subsequently to Oryza sativa plants.

Example 45 Plant Transformation Rice Transformation

The Agrobacterium containing the expression vector was used to transformOryza sativa plants. Mature dry seeds of the rice japonica cultivarNipponbare were dehusked. Sterilization was carried out by incubatingfor one minute in 70% ethanol, followed by 30 minutes in 0.2% HgCl₂,followed by a 6 times 15 minutes wash with sterile distilled water. Thesterile seeds were then germinated on a medium containing 2,4-D (callusinduction medium). After incubation in the dark for four weeks,embryogenic, scutellum-derived calli were excised and propagated on thesame medium. After two weeks, the calli were multiplied or propagated bysubculture on the same medium for another 2 weeks. Embryogenic calluspieces were sub-cultured on fresh medium 3 days before co-cultivation(to boost cell division activity).

Agrobacterium strain LBA4404 containing the expression vector was usedfor co-cultivation. Agrobacterium was inoculated on AB medium with theappropriate antibiotics and cultured for 3 days at 28° C. The bacteriawere then collected and suspended in liquid co-cultivation medium to adensity (OD₆₀₀) of about 1. The suspension was then transferred to aPetri dish and the calli immersed in the suspension for 15 minutes. Thecallus tissues were then blotted dry on a filter paper and transferredto solidified, co-cultivation medium and incubated for 3 days in thedark at 25° C. Co-cultivated calli were grown on 2,4-D-containing mediumfor 4 weeks in the dark at 28° C. in the presence of a selection agent.During this period, rapidly growing resistant callus islands developed.After transfer of this material to a regeneration medium and incubationin the light, the embryogenic potential was released and shootsdeveloped in the next four to five weeks. Shoots were excised from thecalli and incubated for 2 to 3 weeks on an auxin-containing medium fromwhich they were transferred to soil. Hardened shoots were grown underhigh humidity and short days in a greenhouse.

Approximately 35 independent T0 rice transformants were generated forone construct. The primary transformants were transferred from a tissueculture chamber to a greenhouse. After a quantitative PCR analysis toverify copy number of the T-DNA insert, only single copy transgenicplants that exhibit tolerance to the selection agent were kept forharvest of T1 seed. Seeds were then harvested three to five months aftertransplanting. The method yielded single locus transformants at a rateof over 50% (Aldemita and Hodges 1996, Chan et al. 1993, Hiei et al.1994).

Corn Transformation

Transformation of maize (Zea mays) is performed with a modification ofthe method described by Ishida et al. (1996) Nature Biotech 14(6):745-50. Transformation is genotype-dependent in corn and only specificgenotypes are amenable to transformation and regeneration. The inbredline A188 (University of Minnesota) or hybrids with A188 as a parent aregood sources of donor material for transformation, but other genotypescan be used successfully as well. Ears are harvested from corn plantapproximately 11 days after pollination (DAP) when the length of theimmature embryo is about 1 to 1.2 mm. Immature embryos are cocultivatedwith Agrobacterium tumefaciens containing the expression vector, andtransgenic plants are recovered through organogenesis. Excised embryosare grown on callus induction medium, then maize regeneration medium,containing the selection agent (for example imidazolinone but variousselection markers can be used). The Petri plates are incubated in thelight at 25° C. for 2-3 weeks, or until shoots develop. The green shootsare transferred from each embryo to maize rooting medium and incubatedat 25° C. for 2-3 weeks, until roots develop. The rooted shoots aretransplanted to soil in the greenhouse. T1 seeds are produced fromplants that exhibit tolerance to the selection agent and that contain asingle copy of the T-DNA insert.

Wheat Transformation

Transformation of wheat is performed with the method described by Ishidaet al. (1996) Nature Biotech 14(6): 745-50. The cultivar Bobwhite(available from CIMMYT, Mexico) is commonly used in transformation.Immature embryos are co-cultivated with Agrobacterium tumefacienscontaining the expression vector, and transgenic plants are recoveredthrough organogenesis. After incubation with Agrobacterium, the embryosare grown in vitro on callus induction medium, then regeneration medium,containing the selection agent (for example imidazolinone but variousselection markers can be used). The Petri plates are incubated in thelight at 25° C. for 2-3 weeks, or until shoots develop. The green shootsare transferred from each embryo to rooting medium and incubated at 25°C. for 2-3 weeks, until roots develop. The rooted shoots aretransplanted to soil in the greenhouse. T1 seeds are produced fromplants that exhibit tolerance to the selection agent and that contain asingle copy of the T-DNA insert.

Soybean Transformation

Soybean is transformed according to a modification of the methoddescribed in the Texas A&M U.S. Pat. No. 5,164,310. Several commercialsoybean varieties are amenable to transformation by this method. Thecultivar Jack (available from the Illinois Seed foundation) is commonlyused for transformation. Soybean seeds are sterilised for in vitrosowing. The hypocotyl, the radicle and one cotyledon are excised fromseven-day old young seedlings. The epicotyl and the remaining cotyledonare further grown to develop axillary nodes. These axillary nodes areexcised and incubated with Agrobacterium tumefaciens containing theexpression vector. After the cocultivation treatment, the explants arewashed and transferred to selection media. Regenerated shoots areexcised and placed on a shoot elongation medium. Shoots no longer than 1cm are placed on rooting medium until roots develop. The rooted shootsare transplanted to soil in the greenhouse. T1 seeds are produced fromplants that exhibit tolerance to the selection agent and that contain asingle copy of the T-DNA insert.

Rapeseed/Canola Transformation

Cotyledonary petioles and hypocotyls of 5-6 day old young seedling areused as explants for tissue culture and transformed according to Babicet al. (1998, Plant Cell Rep 17: 183-188). The commercial cultivarWestar (Agriculture Canada) is the standard variety used fortransformation, but other varieties can also be used. Canola seeds aresurface-sterilized for in vitro sowing. The cotyledon petiole explantswith the cotyledon attached are excised from the in vitro seedlings, andinoculated with Agrobacterium (containing the expression vector) bydipping the cut end of the petiole explant into the bacterialsuspension. The explants are then cultured for 2 days on MSBAP-3 mediumcontaining 3 mg/l BAP, 3% sucrose, 0.7% Phytagar at 23° C., 16 hr light.After two days of co-cultivation with Agrobacterium, the petioleexplants are transferred to MSBAP-3 medium containing 3 mg/l BAP,cefotaxime, carbenicillin, or timentin (300 mg/l) for 7 days, and thencultured on MSBAP-3 medium with cefotaxime, carbenicillin, or timentinand selection agent until shoot regeneration. When the shoots are 5-10mm in length, they are cut and transferred to shoot elongation medium(MSBAP-0.5, containing 0.5 mg/l BAP). Shoots of about 2 cm in length aretransferred to the rooting medium (MS0) for root induction. The rootedshoots are transplanted to soil in the greenhouse. T1 seeds are producedfrom plants that exhibit tolerance to the selection agent and thatcontain a single copy of the T-DNA insert.

Alfalfa Transformation

A regenerating clone of alfalfa (Medicago sativa) is transformed usingthe method of (McKersie et al., 1999 Plant Physiol 119: 839-847).Regeneration and transformation of alfalfa is genotype dependent andtherefore a regenerating plant is required. Methods to obtainregenerating plants have been described. For example, these can beselected from the cultivar Rangelander (Agriculture Canada) or any othercommercial alfalfa variety as described by Brown D C W and A Atanassov(1985. Plant Cell Tissue Organ Culture 4: 111-112). Alternatively, theRA3 variety (University of Wisconsin) has been selected for use intissue culture (Walker et al., 1978 Am J Bot 65:654-659). Petioleexplants are cocultivated with an overnight culture of Agrobacteriumtumefaciens C58C1 pMP90 (McKersie et al., 1999 Plant Physiol 119:839-847) or LBA4404 containing the expression vector. The explants arecocultivated for 3 d in the dark on SH induction medium containing 288mg/L Pro, 53 mg/L thioproline, 4.35 g/L K2SO4, and 100 μmacetosyringinone. The explants are washed in half-strengthMurashige-Skoog medium (Murashige and Skoog, 1962) and plated on thesame SH induction medium without acetosyringinone but with a suitableselection agent and suitable antibiotic to inhibit Agrobacterium growth.After several weeks, somatic embryos are transferred to BOi2Ydevelopment medium containing no growth regulators, no antibiotics, and50 g/L sucrose. Somatic embryos are subsequently germinated onhalf-strength Murashige-Skoog medium. Rooted seedlings were transplantedinto pots and grown in a greenhouse. T1 seeds are produced from plantsthat exhibit tolerance to the selection agent and that contain a singlecopy of the T-DNA insert.

Example 46 Evaluation Procedure 46.1 Evaluation Setup

Approximately 35 independent T0 rice transformants were generated. Theprimary transformants were transferred from a tissue culture chamber toa greenhouse for growing and harvest of T1 seed. Seven events, of whichthe T1 progeny segregated 3:1 for presence/absence of the transgene,were retained. For each of these events, approximately 10 T1 seedlingscontaining the transgene (hetero- and homo-zygotes) and approximately 10T1 seedlings lacking the transgene (nullizygotes) were selected bymonitoring visual marker expression. The transgenic plants and thecorresponding nullizygotes were grown side-by-side at random positions.Greenhouse conditions were of shorts days (12 hours light), 28° C. inthe light and 22° C. in the dark, and a relative humidity of 70%.

Five T1 events were further evaluated in the T2 generation following thesame evaluation procedure as for the T1 generation but with moreindividuals per event. From the stage of sowing until the stage ofmaturity the plants were passed several times through a digital imagingcabinet. At each time point digital images (2048×1536 pixels, 16 millioncolours) were taken of each plant from at least 6 different angles.

The early vigour is the plant (seedling) aboveground area three weekspost-germination. Increase in root biomass is expressed as an increasein total root biomass (measured as maximum biomass of roots observedduring the lifespan of a plant); or as an increase in the root/shootindex (measured as the ratio between root mass and shoot mass in theperiod of active growth of root and shoot).

46.2 Statistical Analysis: F-Test

A two factor ANOVA (analysis of variants) was used as a statisticalmodel for the overall evaluation of plant phenotypic characteristics. AnF-test was carried out on all the parameters measured of all the plantsof all the events transformed with the gene of the present invention.The F-test was carried out to check for an effect of the gene over allthe transformation events and to verify for an overall effect of thegene, also known as a global gene effect. The threshold for significancefor a true global gene effect was set at a 5% probability level for theF-test. A significant F-test value points to a gene effect, meaning thatit is not only the mere presence or position of the gene that is causingthe differences in phenotype.

To check for an effect of the genes within an event, i.e., for aline-specific effect, a t-test was performed within each event usingdata sets from the transgenic plants and the corresponding null plants.“Null plants” or “null segregants” or “nullizygotes” are the plantstreated in the same way as the transgenic plant, but from which thetransgene has segregated. Null plants can also be described as thehomozygous negative transformed plants. The threshold for significancefor the t-test is set at 10% probability level. The results for someevents can be above or below this threshold. This is based on thehypothesis that a gene might only have an effect in certain positions inthe genome, and that the occurrence of this position-dependent effect isnot uncommon. This kind of gene effect is also named herein a “lineeffect of the gene”. The p-value is obtained by comparing the t-value tothe t-distribution or alternatively, by comparing the F-value to theF-distribution. The p-value then gives the probability that the nullhypothesis (i.e., that there is no effect of the transgene) is correct.

Example 47 Evaluation Results

The plant aboveground area (or leafy biomass) was determined by countingthe total number of pixels on the digital images from aboveground plantparts discriminated from the background. This value was averaged for thepictures taken on the same time point from the different angles and wasconverted to a physical surface value expressed in square mm bycalibration. Experiments show that the aboveground plant area measuredthis way correlates with the biomass of plant parts above ground. Theabove ground area is the time point at which the plant had reached itsmaximal leafy biomass.

The mature primary panicles were harvested, counted, bagged,barcode-labeled and then dried for three days in an oven at 37° C. Thepanicles were then threshed and all the seeds were collected andcounted. The filled husks were separated from the empty ones using anair-blowing device. The empty husks were discarded and the remainingfraction was counted again. The filled husks were weighed on ananalytical balance. The number of filled seeds was determined bycounting the number of filled husks that remained after the separationstep. The total seed yield was measured by weighing all filled husksharvested from a plant. Total seed number per plant was measured bycounting the number of husks harvested from a plant. Thousand kernelweight (TKW) is extrapolated from the number of filled seeds counted andtheir total weight. The harvest index (HI) in the present invention isdefined as the ratio between the total seed yield and the above groundarea (mm²), multiplied by a factor 10⁶. The total number of flowers perpanicle as defined in the present invention is the ratio between thetotal number of seeds and the number of mature primary panicles. Theseed fill rate as defined in the present invention is the proportion(expressed as a %) of the number of filled seeds over the total numberof seeds (or florets).

As presented in Tables Q to W, the aboveground biomass, the number offlowers per panicle, the seed yield, the total number of seeds, thenumber of filled seeds, the thousand kernel weight (TKW) and harvestindex are increased in the transgenic plants with increased expression anucleic acid encoding a SPL15 transcription factor polypeptide, comparedto suitable control plants. Results from the T1 and the T2 generationsare shown.

Table Q shows the number of transgenic events with an increase inaboveground biomass, the percentage of this increase, as well as thestatistical relevance of this increase according to the F-test.

TABLE Q Number of transgenic events with an increase in abovegroundbiomass, the percentage of the increase, and P value of the F-test in T1and T2 generation of transgenic rice with increased expression of anucleic acid encoding an SPL15 transcription factor polypeptide.Aboveground biomass Number of events showing an increase % Difference Pvalue of F test T1 generation 5 out of 7 5 0.0773 T2 generation 4 out of5 7 0.0172

Table R shows the number of transgenic events with an increase in thetotal number of flowers per panicle, the percentage of this increase, aswell as the statistical relevance of this increase according to theF-test.

TABLE R Number of transgenic events with an increase in flowers perpanicle, the percentage of the increase, and P value of the F-test in T1and T2 generation of transgenic rice with increased expression of anucleic acid encoding an SPL15 transcription factor polypeptide. Flowersper panicle Number of events showing an increase % Difference P value ofF test T1 generation 6 out of 7 4 0.0447 T2 generation 4 out of 5 100.0013

Table S shows the number of transgenic events with an increase in totalseed yield (total seed weight), the percentage of this increase, as wellas the statistical relevance of this increase according to the F-test.

TABLE S Number of transgenic events with an increase in seed yield, thepercentage of the increase, and P value of the F-test in T1 and T2generation of transgenic rice with increased expression of a nucleicacid encoding an SPL15 transcription factor polypeptide. Seed yieldNumber of events showing an increase % Difference P value of F test T1generation 7 out of 7 15 0.0019 T2 generation 5 out of 5 21 0.0001

Table T shows the number of transgenic events with an increase in thetotal number of seeds, the percentage of this increase, as well as thestatistical relevance of this increase according to the F-test.

TABLE T Number of transgenic events with an increase in total number ofseeds, the percentage of the increase, and P value of the F-test in T1and T2 generation of transgenic rice with increased expression of anucleic acid encoding an SPL15 transcription factor polypeptide. Totalnumber of seeds Number of events showing an increase % Difference Pvalue of F test T1 generation 6 out of 7 6 0.07 T2 generation 4 out of 513 0.0023

Table U shows the number of transgenic events with an increase in thenumber of filled seeds, the percentage of this increase, as well as thestatistical relevance of this increase according to the F-test.

TABLE U Number of transgenic events with an increase in number of filledseeds, the percentage of the increase, and P value of the F-test in T1and T2 generation of transgenic rice with increased expression of anucleic acid encoding an SPL15 transcription factor polypeptide. Numberof filled seeds Number of events showing an increase % Difference Pvalue of F test T1 generation 6 out of 7 14 0.0031 T2 generation 4 outof 5 19 0.0003

Table V shows the number of transgenic events with an increase in theharvest index, the percentage of this increase, as well as thestatistical relevance of this increase according to the F-test.

TABLE V Number of transgenic events with an increase in harvest index,the percentage of the increase, and P value of the F-test in T1 and T2generation of transgenic rice with increased expression of a nucleicacid encoding an SPL15 transcription factor polypeptide. Harvest indexNumber of events showing an increase % Difference P value of F test T1generation 7 out of 7 12 0.0003 T2 generation 4 out of 5 15 0.0001

Table W shows the number of transgenic events with an increase in thethousand kernel weight (TKW), the percentage of this increase, as wellas the statistical relevance of this increase according to the F-test.

TABLE W Number of transgenic events with an increase in thousand kernelweight (TKW), the percentage of the increase, and P value of the F-testin T1 and T2 generation of transgenic rice with increased expression ofa nucleic acid encoding an SPL15 transcription factor polypeptide.Thousand kernel weight Number of events showing an increase % DifferenceP value of F test T1 generation 4 out of 7 2 0.0041 T2 generation 4 outof 5 1 0.0259

Example 48 Results of the Phenotypic Evaluation of the Transgenic PlantsExpressing SEQ ID NO: 234 Under the Control of a Constitutive Promoter

The results of the evaluation of transgenic rice plants expressing thenucleic acid sequence useful in performing the methods of the invention,under the control of the constitutive GOS2 promoter, are presented inTable X. The percentage difference between the transgenics and thecorresponding nullizygotes is also shown.

The transgenic plants expressing the nucleic acid sequence useful inperforming the methods of the invention, have increased early vigour andincreased TKW compared to the control plants (in this case, thenullizygotes).

TABLE X Results of the evaluation of T1 generation transgenic riceplants expressing the nucleic acid sequence useful in performing themethods of the invention, under the control of a strong constitutiveGOS2 promoter. % Increase of the best events Trait in T1 generationIncreased early vigour 17% Total seed yield (per plant) 18% Total numberof filled seeds 19% Increased seed fill rate 10% Increased harvest index15%

1-171. (canceled)
 172. A method for enhancing yield-related traits in aplant relative to suitable control plants, comprising preferentiallyreducing expression of an endogenous MADS15 gene in said plant.
 173. Themethod of claim 172, wherein said MADS15 gene encodes a protein thatcomprises any one or more of the following motifs: SEQ ID NO: 114, SEQID NO: 115, SEQ ID NO: 116, and SEQ ID NO:
 117. 174. The method of claim172, wherein said reduced expression is effected by RNA-mediateddownregulation of gene expression.
 175. The method of claim 174, whereinsaid RNA-mediated downregulation is effected by co-suppression.
 176. Themethod of claim 174, wherein said RNA-mediated downregulation iseffected by use of antisense MADS15 nucleic acid sequences.
 177. Themethod of claim 172, wherein said reduced expression is effected usingan inverted repeat of a MADS15 gene or fragment thereof, preferablycapable of forming a hairpin structure.
 178. The method of claim 172,wherein said reduced expression is effected using ribozymes withspecificity for a MADS15 nucleic acid.
 179. The method of claim 172,wherein said reduced expression is effected by insertion mutagenesis.180. The method of claim 172, wherein said endogenous MADS15 gene is aMADS15 gene found in a plant in its natural form or is an isolatedMADS15 nucleic acid subsequently introduced into a plant.
 181. Themethod of claim 180, wherein said isolated MADS15 nucleic acidsubsequently introduced into a plant is operably linked to aconstitutive promoter or a GOS2 promoter.
 182. The method of claim 180,wherein said isolated MADS15 nucleic acid subsequently introduced into aplant is (a) derived from a plant source or artificial source, (b)homologous to the endogenous MADS15 gene, (c) derived from amonocotyledonous plant and is used for transformation of amonocotyledonous plant, (d) derived from the family Poaceae and is usedfor transformation of a plant of the family Poaceae, or (e) derived fromrice and is used to transform a rice plant.
 183. The method of claim182, wherein said MADS15 nucleic acid sequence from rice comprises asufficient length of substantially contiguous nucleotides of SEQ ID NO:109 (OsMADS15) or comprises a sufficient length of substantiallycontiguous nucleotides of a nucleic acid sequence encoding an orthologueor paralogue of OsMADS15 (SEQ ID NO: 110).
 184. The method of claim 183,wherein said orthologues or paralogues of OsMADS15 are represented bySEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ IDNO: 155, SEQ ID NO: 157, SEQ ID NO: 159 and SEQ ID NO:
 161. 185. Themethod of claim 183, wherein said substantially contiguous nucleotidesof a nucleic acid sequence encoding an orthologue or paralogue ofOsMADS15 (SEQ ID NO: 110) are substantially contiguous nucleotides fromnucleic acid sequences represented by SEQ ID NO: 146, SEQ ID NO: 148,SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ IDNO: 158 and SEQ ID NO:
 160. 186. The method of claim 172, wherein saidenhanced yield related traits comprises increased yield, preferablyincreased seed yield and/or increased biomass.
 187. The method of claim186, wherein said increased seed yield is selected from one or more ofthe following: (a) increased seed biomass (seed weight), (b) increasednumber of flowers per plant, and (c) increased number of (filled) seeds.188. A plant or part thereof, including seeds, obtained by the method ofclaim 172, wherein said plant or plant part comprises a recombinantMADS15 nucleic acid.
 189. A construct comprising: (a) a MADS15 nucleicacid capable of silencing an endogenous MADS15 gene; (b) one or morecontrol sequences capable of driving expression of the nucleic acidsequence of (a); and optionally (c) a transcription terminationsequence.
 190. The construct of claim 189, wherein said one or morecontrol sequences is at least a constitutive promoter or a GOS2promoter.
 191. A method of producing a plant having enhancedyield-related traits relative to control plants, comprising transformingthe construct of claim 189 into a plant, wherein the enhancedyield-related traits are increased seed yield and/or biomass.
 192. Aplant, plant part or plant cell transformed with the construct of claim189.
 193. A method for the production of a transgenic plant havingenhanced yield related traits relative to control plants, which methodcomprises: (a) introducing and expressing in a plant a MADS15 nucleicacid capable of silencing an endogenous MADS15 gene; and (b) cultivatingthe plant cell under condition promoting plant growth and development.194. A transgenic plant having enhanced yield related traits relative tocontrol plants, said enhanced yield related traits resulting fromdecreased expression of a nucleic acid encoding a MADS15 protein,wherein the amino acid sequence of said MADS15 protein comprises any oneor more of the following motifs: SEQ ID NO: 114, SEQ ID NO: 115, SEQ IDNO: 116, and SEQ ID NO: 117; or a transgenic plant cell derived fromsaid transgenic plant.
 195. The transgenic plant or part thereof ofclaim 188, wherein said plant is a crop plant, a monocot or a cereal,such as rice, maize, wheat, barley, millet, rye, triticale, sorghum andoats, or a transgenic plant cell derived from said transgenic plant.196. Harvestable parts of the plant of claim 195, wherein saidharvestable parts are preferably seeds.
 197. Products derived from theplant of claim 195 and/or from the harvestable parts of said plant. 198.The transgenic plant, plant part or plant cell of claim 192, whereinsaid plant is a crop plant, a monocot or a cereal, such as rice, maize,wheat, barley, millet, rye, triticale, sorghum and oats, or a transgenicplant cell derived from said transgenic plant.
 199. Harvestable parts ofthe plant of claim 198, wherein said harvestable parts are preferablyseeds, or products derived from said plant and/or from said harvestableparts.
 200. The transgenic plant, plant part or plant cell of claim 194,wherein said plant is a crop plant, a monocot or a cereal, such as rice,maize, wheat, barley, millet, rye, triticale, sorghum and oats, or atransgenic plant cell derived from said transgenic plant. 201.Harvestable parts of the plant of claim 200, wherein said harvestableparts are preferably seeds, or products derived from said plant and/orfrom said harvestable parts.